WO2022126416A1 - 抗bcma抗体及其制备方法和应用 - Google Patents

抗bcma抗体及其制备方法和应用 Download PDF

Info

Publication number
WO2022126416A1
WO2022126416A1 PCT/CN2020/136748 CN2020136748W WO2022126416A1 WO 2022126416 A1 WO2022126416 A1 WO 2022126416A1 CN 2020136748 W CN2020136748 W CN 2020136748W WO 2022126416 A1 WO2022126416 A1 WO 2022126416A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
bcma
variant
amino acid
Prior art date
Application number
PCT/CN2020/136748
Other languages
English (en)
French (fr)
Inventor
方丽娟
石剑
华珊
张敬
张凯莉
周鹏飞
Original Assignee
武汉友芝友生物制药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 武汉友芝友生物制药股份有限公司 filed Critical 武汉友芝友生物制药股份有限公司
Priority to US18/257,749 priority Critical patent/US20240117061A1/en
Priority to PCT/CN2020/136748 priority patent/WO2022126416A1/zh
Priority to CN202080107895.2A priority patent/CN116783220A/zh
Publication of WO2022126416A1 publication Critical patent/WO2022126416A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of medicine, in particular to the antibody of B cell maturation antigen (also called B Cell Maturation Antigen, BCMA) and its preparation method and application.
  • B cell maturation antigen also called B Cell Maturation Antigen, BCMA
  • MM Multiple myeloma
  • SMM smokeless MM
  • active MM active MM
  • PCL plasma cell leukemia
  • the global incidence rate is 114,000 person-times/year, showing an increasing trend year by year, and the fatality rate is 80,000 person-times/year.
  • the most common age group is 65-74 years, and complications include hypercalcemia, renal insufficiency, anemia, and infection.
  • the main methods of treating MM include autologous stem cell transplantation, combined with traditional chemotherapy drugs, immunomodulatory drugs (including thalidomide, lenalidomide and pomalidomide), and proteasome inhibitors (including bortezomib, carfilzomib and ixazomib).
  • immunomodulatory drugs including thalidomide, lenalidomide and pomalidomide
  • proteasome inhibitors including bortezomib, carfilzomib and ixazomib.
  • Therapeutic mAbs can effectively bind to effector cells expressing Fc ⁇ receptors through their specialized Fc, inducing effector cells to produce antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity ( complement-dependent cytotoxicity, CDC) and antibody-dependent cell-mediated endocytosis to clear MM cells.
  • ADCC antibody-dependent cell cytotoxicity
  • CDC complement-dependent cytotoxicity
  • antibody-dependent cell-mediated endocytosis to clear MM cells.
  • the U.S. FDA approved two therapeutic monoclonal antibodies, Daratumumab targeting CD38 and Elotuzumab targeting SLAMF7. Due to their low toxicity profile, they can be used in combination with existing therapies in new-onset MM patients and RRMM patients with clinical benefits. obvious.
  • BCMA tumor necrosis factor receptor superfamily member 17
  • TNFRSF17 tumor necrosis factor receptor superfamily member 17
  • BCMA B-cell maturation antigen
  • CD269 CD269
  • BAFF-R B-cell activation factor receptor
  • TACI transmembrane activator and calcium modulator and cyclophilin ligand interactor
  • BCMA is specifically highly expressed in MM tissues and cells, and is associated with the disease progression of MM.
  • BAFF-R was almost undetectable in MM cell lines or patient MM cells, and the positive ratio and expression intensity of TACI were weaker than those of BCMA.
  • overexpression of BCMA can activate downstream AKT, MAPK, NF ⁇ B signaling pathways, induce key anti-apoptotic proteins Mcl1, Bcl2, Bcl-xL, pro-microangiogenic protein CD31 and vascular endothelial cells Expression of growth factor VEGF.
  • BCMA multiple myeloma
  • the present invention provides a specific monoclonal antibody against BCMA.
  • the present invention relates to the following technical solutions:
  • An anti-BCMA antibody or an antigen-binding fragment thereof characterized in that it comprises
  • HCDR1, HCDR2 and HCDR3 contained in the variable region of the heavy chain set forth in SEQ ID NO: 3; and/or LCDR1, LCDR2 and LCDR3 contained in the variable region of the light chain set forth in SEQ ID NO: 4:
  • HCDR1, HCDR2 and HCDR3 contained in the variable region of the heavy chain set forth in SEQ ID NO: 5; and/or LCDR1, LCDR2 and LCDR3 contained in the variable region of the light chain set forth in SEQ ID NO: 6: or
  • the anti-BCMA antibody comprises the following CDRs,
  • HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof
  • HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof,
  • HCDR3 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof,
  • LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof
  • LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof
  • LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof;
  • HCDR1 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 10 or a variant thereof
  • HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof,
  • HCDR3 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof,
  • LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof
  • LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof
  • LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof;
  • HCDR1 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof
  • HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof,
  • HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 15 or a variant thereof
  • LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof
  • LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof
  • LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof
  • the variant has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% of the corresponding CDR sequence shown in the sequence number , preferably a sequence of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and retaining binding affinity to BCMA, or to the corresponding CDRs indicated by SEQ ID NO:
  • An amino acid sequence that has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence and retains binding affinity to BCMA;
  • the heavy chain variable region and/or light chain variable region of the antibody or antigen-binding fragment thereof comprises FR regions from human, murine or rabbit origin.
  • SEQ ID NO: 3 SEQ ID NO: 5, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30; and
  • the light chain variable region of the antibody comprises or consists of the following sequences or variants thereof: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: : 25, SEQ ID NO: 27, SEQ ID NO: 29 or SEQ ID NO: 31;
  • the variant has at least 80%, 81%, 82%, 83%, 84%, 85%, 86% of the amino acid sequence of the corresponding antibody heavy chain variable region or light chain variable region shown in SEQ ID NO: %, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and retain binding to BCMA Affinity sequence, or has one or more (preferably 1, 2, 3, 4, 5, 6, 7) compared with the amino acid sequence of the corresponding antibody heavy chain variable region or light chain variable region shown in SEQ ID NO: , 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) and amino acid sequences that retain binding affinity to BCMA.
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 1 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 2 or a variant thereof ;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 3 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 4 or a variant thereof ;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 5 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 6 or a variant thereof ;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 24 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 25 or a variant thereof ;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 26 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 27 or a variant thereof ;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 28 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 29 or a variant thereof ;
  • the amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 30 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 31 or a variant thereof .
  • anti-BCMA antibody or antigen-binding fragment thereof wherein the antigen-binding fragment is selected from Fab, Fab', F(ab') 2 , F(ab) 2 , Fd, Fv, dAb, Fab /c, complementarity determining region fragment, scFv, scFv multimer, disulfide bond stabilization Fv (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), diabody, disulfide Bond-stabilized diabodies (ds-Diabodies), multispecific antibodies, single domain antibodies (sdabs), Nanobodies, domain antibodies or bivalent domain antibodies formed from a portion of an antibody comprising one or more CDRs.
  • the antigen-binding fragment is selected from Fab, Fab', F(ab') 2 , F(ab) 2 , Fd, Fv, dAb, Fab /c, complementarity determining region fragment,
  • An isolated polypeptide or variant thereof comprising the sequences shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, wherein the polypeptide, as part of an anti-BCMA antibody, specifically binds to BCMA , the antibody also comprises the sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18;
  • An isolated polypeptide or variant thereof comprising the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, wherein the polypeptide, as part of an anti-BCMA antibody, specifically binds to BCMA , the antibody also comprises the sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21;
  • An isolated polypeptide or a variant thereof comprising the sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, wherein the polypeptide, as part of an anti-BCMA antibody, specifically binds to BCMA , the antibody also comprises the sequences shown in SEQ ID NO: 22, SEQ ID NO: 17 and SEQ ID NO: 23;
  • An isolated polypeptide or a variant thereof comprising the sequence shown in SEQ ID NO: 1, wherein the polypeptide, as part of an anti-BCMA antibody, specifically binds to BCMA, and the antibody further comprises the sequence shown in SEQ ID NO: 2 the sequence shown;
  • An isolated polypeptide or a variant thereof comprising the sequence shown in SEQ ID NO: 2, wherein the polypeptide is used as a part of an anti-BCMA antibody that specifically binds to BCMA, and the antibody also comprises the sequence shown in SEQ ID NO: 1 the sequence shown;
  • polypeptide or a variant thereof comprising the sequence shown in SEQ ID NO: 3, wherein the polypeptide, as part of an anti-BCMA antibody, specifically binds to BCMA, and the antibody further comprises the sequence shown in SEQ ID NO: 4 the sequence shown;
  • polypeptide or a variant thereof comprising the sequence shown in SEQ ID NO: 4, wherein the polypeptide, as a part of an anti-BCMA antibody, specifically binds to BCMA, and the antibody further comprises the sequence shown in SEQ ID NO: 3 the sequence shown;
  • polypeptide or a variant thereof comprising the sequence shown in SEQ ID NO: 5, wherein the polypeptide, as part of an anti-BCMA antibody, specifically binds to BCMA, and the antibody further comprises the sequence shown in SEQ ID NO: 6 the sequence shown;
  • An isolated polypeptide or a variant thereof comprising the sequence shown in SEQ ID NO: 6, wherein the polypeptide, as part of an anti-BCMA antibody, specifically binds to BCMA, and the antibody further comprises the sequence shown in SEQ ID NO: 5 the sequence shown;
  • polypeptide or a variant thereof comprising the sequence shown in SEQ ID NO: 24, wherein the polypeptide is used as a part of an anti-BCMA antibody that specifically binds to BCMA, and the antibody further comprises the sequence shown in SEQ ID NO: 25 the sequence shown;
  • polypeptide or a variant thereof comprising the sequence shown in SEQ ID NO: 25, wherein the polypeptide is used as a part of an anti-BCMA antibody that specifically binds to BCMA, and the antibody further comprises the sequence shown in SEQ ID NO: 24 the sequence shown;
  • An isolated polypeptide or a variant thereof comprising the sequence shown in SEQ ID NO: 26, wherein the polypeptide, as part of an anti-BCMA antibody, specifically binds to BCMA, the antibody further comprising the sequence shown in SEQ ID NO: 27 the sequence shown;
  • An isolated polypeptide or a variant thereof comprising the sequence shown in SEQ ID NO: 28, wherein the polypeptide is used as a part of an anti-BCMA antibody that specifically binds to BCMA, and the antibody further comprises the sequence shown in SEQ ID NO: 29 the sequence shown;
  • variant is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% of the corresponding sequence shown in the sequence number, preferably Sequences that are at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence and retain binding affinity to BCMA, or compared to the corresponding sequence indicated by SEQ ID NO:
  • An amino acid sequence that has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) and retains binding affinity to BCMA.
  • a vector comprising the nucleic acid molecule of item 8.
  • a host cell comprising the nucleic acid molecule of item 8, or the vector of item 9.
  • a conjugate comprising the antibody or antigen-binding fragment thereof described in any one of items 1-6 and a coupling moiety, wherein the coupling moiety is a purification tag (such as a His tag), a detectable tag, Drugs, toxins, cytokines, enzymes, or combinations thereof; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, a chemotherapeutic agent, a biotoxin, polyethylene glycol, or an enzyme.
  • the coupling moiety is a purification tag (such as a His tag), a detectable tag, Drugs, toxins, cytokines, enzymes, or combinations thereof; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, a chemotherapeutic agent, a biotoxin, polyethylene glycol, or an enzyme.
  • a fusion protein or a multispecific antibody comprising the antibody or antigen-binding fragment thereof described in any one of items 1-6; preferably, the fusion protein is a CAR construct, and the The CAR construct specifically binds BCMA.
  • a kit comprising the antibody or antigen-binding fragment thereof described in any one of items 1-6, the conjugate described in item 11 or the fusion protein or multispecific antibody described in item 12; preferably, the The kit also includes a second antibody that specifically recognizes the antibody; optionally, the second antibody further includes a detectable label, such as a radioisotope, fluorescent substance, chemiluminescent substance, colored substance or enzyme; preferably Typically, the kit is used to detect the presence or level of BCMA in a sample.
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of items 1-6, the conjugate of item 11 or the fusion protein or multispecific antibody of item 12; optionally , the pharmaceutical composition also includes a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition is suitable for subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection The form of administration by injection.
  • the antibody or antigen-binding fragment thereof of any one of items 1-6, the conjugate of item 11, or the fusion protein or multispecific antibody of item 12 are used for the treatment and/or prevention of tumors (e.g. multiple myeloma), or in the manufacture of a medicament for the treatment and/or prevention of tumors (eg, multiple myeloma), or in the manufacture of a medicament for the diagnosis of tumors.
  • tumors e.g. multiple myeloma
  • a medicament for the treatment and/or prevention of tumors e.g. multiple myeloma
  • a kit comprising (1) the antibody or antigen-binding fragment thereof of any one of items 1-6, the conjugate of item 11 or the fusion protein or multispecific antibody of item 12, and (2) Antibodies or antigen-binding fragments thereof to other antigens (eg, CD38 and/or SLAMF7), and/or cytotoxic agents, and optionally, instructions for use.
  • antigens eg, CD38 and/or SLAMF7
  • a method for treating or preventing tumors comprising administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of items 1-6, the conjugate of item 11 or the fusion protein or multispecific antibody of item 12.
  • the CDR regions of an antibody are responsible for the binding specificity of the antibody to an antigen.
  • the known sequences of antibody heavy and light chain variable regions there are currently several methods for determining antibody CDR regions, including the Kabat, IMGT, Chothia and AbM numbering systems.
  • the application of each definition with respect to the CDRs of an antibody or variant thereof will be within the scope of the terms as defined and used herein.
  • Given the variable region amino acid sequence of the antibody one skilled in the art can generally determine which residues comprise a particular CDR, without relying on any experimental data other than the sequence itself.
  • antibody or “antigen-binding fragment” refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.
  • the term “antibody” is used in a broad sense and includes immunoglobulin or antibody molecules including monoclonal or polyclonal human, humanized, complex and chimeric antibodies and antibody fragments. Antibodies can be whole antibodies or any antibody fragment, antigen-binding fragment, or single chain thereof.
  • the term “antibody” thus includes any protein or peptide that contains a molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
  • the immunoglobulin molecules or antibody molecules of the present application can be of any class (eg, IgG, IgE, IgM, IgD, IgA, and IgY), of any class of immunoglobulin molecules (eg, IgGl, IgG2, IgG3, IgG4, IgA1, and IgA2) ) or subclass.
  • antibody fragment or "antigen-binding fragment” includes, but is not limited to, F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, Fd, dAb, Fab/c, complementarity determining regions (CDRs) Fragments, single-chain Fvs (scFv), disulfide-stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), diabodies (Diabody), disulfide Bond-stabilized diabodies (ds-Diabodies), scFv multimers (eg, scFv dimers, scFv trimers), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, Nanobodies, Single domain antibody (sdab), domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain the entire antibody structure.
  • CDRs complementarity
  • an antigen-binding fragment includes any polypeptide or complex of polypeptides capable of binding the same antigen to which the parent antibody or parent antibody fragment binds.
  • dsFv disulfide stabilized Fv Fragments
  • Progress in Biochemistry and Biophysics, 1998, 25(6):525-526 describes the structure of dsFv.
  • Holt et al. "Domain antibodies: proteins for therapy” Trends in Biotechnology (2003): Vol. 21, No. 11: 484-490 review antigen-binding fragments called "domain antibodies” or dAbs, which contain only the VH or The VL domain is therefore smaller than eg Fab and scFv.
  • dAbs are the smallest known antigen-binding fragments of antibodies, ranging from 11 kDa to 15 kDa.
  • the term “antibody fragment” includes aptamers, aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer
  • Embodiments of the present application provide various anti-BCMA antibodies comprising at least one antigen binding domain targeting a BCMA antigen.
  • Antigen binding domains that bind BCMA antigen are Fab, or ScFv, or non-covalent pairing (Fv) between variable heavy chain (VH)-light chain variable region (VL).
  • VH variable heavy chain
  • VL variable chain variable region
  • Any of the above-described antibodies or polypeptides may also include additional polypeptides, eg, a signal peptide at the N-terminus of the antibody for directing secretion, or other heterologous polypeptides as described herein.
  • the present invention includes not only complete antibodies, but also immunologically active antibody fragments or fusion proteins formed by antibodies and other sequences.
  • the present invention also provides other protein or fusion expression products with the antibodies of the present invention.
  • the present invention includes any proteins or protein conjugates and fusion expression products (ie, immunoconjugates and fusion expression products) having variable region-containing heavy and light chains, as long as the variable region is compatible with the antibody of the invention
  • the variable regions of the heavy and light chains are identical or at least 90% homologous, preferably at least 95% homologous. Therefore, the present invention includes those molecules having CDR-bearing monoclonal antibody light and heavy chain variable regions, as long as their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) of the CDRs of the present invention homology.
  • the invention also includes fragments, variants, derivatives and analogs of the antibodies.
  • Antibodies, antigen-binding fragments, variants or derivatives thereof of the present application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies (eg, bispecific antibodies, trispecific antibodies, etc.), human antibodies , animal-derived antibodies, humanized antibodies, primatized (primatized) antibodies, or chimeric antibodies, CDR-grafted and/or modified antibodies, single-chain antibodies (eg, scFv), diabodies, antigen tables
  • Binding fragments e.g., Fab, Fab' and F(ab') 2 , Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (dsFv), comprising VL domains or VH structures Fragments of domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies.
  • Antibody fragments, antigen-binding fragments, derivatives or analogs of the invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substitutions
  • the amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide with a substitution group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as an extended polypeptide half-life) A compound formed by fusion of a compound such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretory sequence or a sequence or protein used to purify the polypeptide). sequence, or a fusion protein with a 6His tag).
  • the antibody of the present invention refers to a polypeptide comprising the above-mentioned CDR region having human BCMA-binding activity.
  • the term also includes variant forms of the polypeptides comprising the above-mentioned CDR regions having the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein.
  • the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency The encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention.
  • the percent "sequence homology" (also referred to herein as "amino acid homology") between a first amino acid sequence and a second amino acid sequence can be determined by using [ Calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], where the first Each deletion, insertion, substitution or addition of amino acid residues in the diamino acid sequence - compared to the first amino acid sequence - is considered to be a difference in a single amino acid residue (position), i.e., considered to be "Amino Acid Differences" as defined.
  • amino acid sequence with the greatest number of amino acid residues is considered the "first" amino acid sequence for purposes of determining the percent "sequence identity" between two amino acid sequences according to the calculation methods listed above, And another amino acid sequence is considered a "second" amino acid sequence.
  • amino acid substitutions can generally be described as amino acid substitutions in which amino acid residues are replaced with similar chemical A substitution of another amino acid residue of a structure that has little or no effect on the function, activity, or other biological property of the polypeptide.
  • Constant amino acid substitutions are those in which amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid) ), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanine , valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), side chains of beta-branched chains (eg, threonine, valine, isoleucine amino acid) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains eglysine, arginine, histidine
  • non-essential amino acid residues of an immunoglobulin polypeptide are preferably replaced by other amino acid residues from the same side chain family.
  • a string of amino acids can be replaced by a structurally similar string of amino acids that differ in sequence and/or composition of side chain families.
  • Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conservative substitution between the two amino acids.
  • the conservative substitution is preferably one in which one amino acid within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) a small Aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) ) Aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative substitutions are as follows: Ala for Gly or Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Gly is replaced by Ala or by Pro; His by Asn or by Gln; Ile by Leu or by Val; Leu by Ile or by Val; Lys by Arg, by Gln or by Glu; Met Replace with Leu, with Tyr or with Ile; Phe with Met, with Leu or with Tyr; Ser with Thr; Thr with Ser; Trp with Tyr; Tyr with Trp; and/or Phe into Val, replace with Ile or replace with Leu.
  • the antibodies of the invention can bind a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.
  • Antibodies of the invention may be linked or fused to therapeutic agents, which may include detectable labels such as radiolabels, immunomodulatory agents, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cells Toxic agents, which may be drugs or toxins, ultrasound enhancers, non-radioactive labels, combinations thereof, and other such ingredients known in the art.
  • the anti-BCMA antibodies of the present invention have, for example, one or more of the following advantages:
  • the antibody of the present invention has excellent biological activity and specificity, and has a high affinity with BCMA, higher binding activity than existing BCMA antibodies (such as 83A10 antibody), and has no obvious potential toxic and side effects.
  • the antibody of the present invention is humanized and still maintains high affinity and reduces immunogenicity.
  • the antibody of the present invention has binding activity to BCMA of cynomolgus monkey, which is convenient for testing in animal models and for quality control detection.
  • the antibody of the present invention has good stability, especially under acidic environment and heat treatment conditions.
  • Figure 1 Shows the expression plasmid map of the corresponding human (A), monkey (B), and mouse (C) full-length BCMA for the construction of stable transfected cell lines HEK293 huBCMA, HEK293 cynoBCMA and HEK293 mBCMA.
  • Figure 2 Shows the BCMA expression identification of human, monkey, and murine BCMA stably transfected cell lines HEK293 huBCMA, HEK293 cynoBCM and HEK293 mBCMA by FACS.
  • Figure 3 Shows the human-monkey crossover detection of antibodies in triplicate hybridoma supernatants of 18F6G3H9, 202E11H10E3 and 211B11H10G5 by FACS.
  • Figure 4 Shows the inhibition of the binding of natural ligand APRIL to human BCMA by competitive ELISA detection of chimeric antibodies c-mAb1, c-mAb2, and c-mAb3.
  • Figure 5 Shows the inhibition of the binding of positive control antibody 83A10 to human BCMA by competitive ELISA detection of chimeric antibodies c-mAb1, c-mAb2, and c-mAb3.
  • Figure 6 Shows the inhibition of the binding of the positive control antibody 83A10 to monkey BCMA by the chimeric antibody c-mAb3 by competitive ELISA.
  • Figure 7 Shows the competitive ELISA detection of humanized antibodies hu-mAb1, hu-mAb2, hu-mAb3, hu-mAb4 inhibition of natural ligand APRIL binding to human BCMA.
  • Figure 8 Shows the competitive ELISA to detect the inhibition of humanized antibodies hu-mAb1, hu-mAb2, hu-mAb3, hu-mAb4 on the binding of positive control antibody 83A10 to human BCMA.
  • Figure 9 Shows the inhibition of the binding of the positive control antibody 83A10 to monkey BCMA by the humanized antibodies hu-mAb3 and hu-mAb4 detected by competitive ELISA.
  • Figure 10 Shows that humanized mAbs hu-mAb1, hu-mAb2, hu-mAb3, hu-mAb4 mediate ADCC effect of effector cell NK92MI-CD16a on target cell NCI-H929.
  • Figure 11 Shows that humanized mAbs hu-mAb1, hu-mAb2, hu-mAb3, hu-mAb4 mediate ADCC effect of effector cell NK92MI-CD16a on target cell U266B1.
  • Recombinant human BCMA-huFc and BCMA-mFc fusion proteins corresponding to amino acids 1 to 54 of human BCMA (SEQ ID NO: 32), hereinafter referred to as huBCMA-huFc and huBCMA-mFc; corresponding to 1 to 53 of cynomolgus monkey BCMA Recombinant cynomolgus monkey BCMA-huFc and BCMA-mFc of amino acids at position 1 (SEQ ID NO: 33), hereinafter referred to as cynoBCMA-huFc and cynoBCMA-mFc; corresponding to amino acids 1 to 49 of mouse BCMA (SEQ ID NO: 34)
  • the recombinant mouse BCMA-huFc and BCMA-mFc fusion proteins hereinafter referred to as mBCMA-huFc and mBCMA-mFc.
  • the above sequence information was obtained from the National Center for Biotechnology Information.
  • the recombinant human hu BCMA-his fusion protein of human BCMA (ACR0Biosystem, Cat. No. BCA-H522y) and the recombinant cynoBCMA-his of monkey BCMA, (ACR0Biosystem, Cat. No. BCA-C52H7) were used for characterization analysis. These materials are used for binding and affinity measurements.
  • the amino acid sequence information of the BCMA ECD molecule is shown in Table 1.
  • Vectors presenting human BCMA Fig. 1A
  • cynomolgus BCMA Fig. 1B
  • mouse BCMA Fig. 1C
  • the green fluorescent protein-positive monoclones were sorted by FACS and cultured, and then anti-human BCMA antibody (Biolegend, Cat. No. 357504), anti-cynomolgus monkey BCMA antibody 83A10 (for the sequence, see the clone No.
  • the shift rates of the stably transfected cell lines HEK293 huBCMA, HEK293 cynoBCMA and HEK293 mBCMA were 93.5%, 92.4% and 98.6%, respectively, indicating that HEK293 huBCMA, HEK293 cynoBCMA and HEK293
  • the mBCMA cell line stably expresses BCMA on the cell surface.
  • the immunization protocol was carried out as follows: BALB/c, C57BL/6, SJL, ICR mice and SD rats (purchased from Southern Model Organisms) were subjected to multiple subcutaneous/peritoneal injections with huBCMA-his fusion as the antigen after emulsification with adjuvant For immunization, monitor the serum titer of the immunized mice/rat, and perform multiple immunizations if the titer is not reached. After meeting the requirements, the spleen cells of the animals were electro-fused with myeloma (Sp2/0) cells.
  • Sp2/0 myeloma
  • the hybridoma polyclonal cells were obtained, and the positive polyclonal cells were screened by ELISA and FACS.
  • the positive clones were subcloned to obtain stable single positive hybridoma cells, and the positive clones were screened by ELISA and FACS.
  • three monoclonal cell lines 18F6G3H9, 202E11H10E3 and 211B11H10G5 with very high binding activity to BCMA were obtained, as shown in Table 2 and FIG. 3 .
  • hybridoma supernatants of these three monoclonal cell lines were sequenced, cloned and expressed, wherein the sequencing results were shown in Example 3, so as to further construct human IgG1 chimeric antibodies and complete the humanization of monoclonal antibodies.
  • the V region sequencing results of the three mouse BCMA antibodies are as follows, and the CDR sequences are shown in Table 3:
  • BCMA antibody in mouse ascites was obtained by protein A affinity chromatography. Purified mouse BCMA antibody concentration was determined by UV absorbance at 280 nm and the corresponding extinction coefficient for each protein. The purity of the antibodies was assessed by SDS-PAGE (all >90% pure), and the endotoxin content of the antibodies was determined using LAL assay (all endotoxin content ⁇ 3 EU/mg).
  • the murine monoclonal antibodies 18F6G3H9, 202E11H10E3 and 211B11H10G5 obtained by purifying the supernatant of the hybridoma were evaluated by ELISA, BiaCore molecular binding assay and flow cytometry.
  • U266B1 and NCI-H929 Binding to cancer cell lines U266B1 and NCI-H929, wherein U266B1 and NCI-H929 were purchased from China Type Culture Center.
  • the purpose of the screening assay is to identify the cross-reactivity of murine antibodies with human BCMA and with cynomolgus BCMA at the molecular and cellular levels.
  • the affinity of the three mouse-derived antibodies to BCMA antigen was evaluated by ELISA, and the affinity and species cross were determined.
  • Test material BCMAECD: huBCMA-huFc and cynoBCMA-huFc; PBS buffer (Phosphate Buffered Saline, pH 74, Gibco, C10010500BT); BSA (Bovogen, BSA0.1), TWEEN 20 (Sinopharm, 30189328 ) and HRP Goat Anti-Mouse IgG (Abclonal, AS003), TMB (BD, 55214), H 2 SO 4 (Sinopharm, 10021618).
  • Test method The BCMA antigens of human, mouse and monkey were respectively prepared into 0.5 ⁇ g/mL coating solution with PBS buffer, 100 ⁇ L/well was added to the ELISA plate, and after coating overnight at 4°C, the residue of the coating plate was discarded, and 3% BSA, 300 ⁇ L per well, was blocked for 3 hours at room temperature. Add 300 ⁇ L of PBST (PBS containing 0.1% TWEEN20) to each well to wash once, three kinds of mouse BCMA antibodies, with 10 ⁇ g/mL as the starting concentration, were diluted 7 times in 7 gradients, and 100 ⁇ L/well was added to the ELISA plate .
  • PBST PBS containing 0.1% TWEEN20
  • the affinity of the three murine antibodies to cell surface BCMA antigen was evaluated by FACS method.
  • Test materials cell line NCI-H929, U266B1, HEK293 cynoBCMA, HEK293; PBS buffer; FBS (Fetal Bovine Serum, Gibco, 10099141); PE Goat anti-mouse IgG Fc (PE Goat anti-mouse IgG Fc) (Biolegend, 405307 ).
  • Test method Resuspend cells with PBS to prepare cell suspension so that the number of cells per well is 200,000. Add 50 ⁇ L of the mouse-derived BCMA antibody to be tested to the 96-well plate, the initial concentration of the antibody is 3000nM, 3-fold serial dilution, and incubate at 4°C for 1 hour. After the primary antibody incubation, add 150 ⁇ L of pre-cooled 1% FBS in PBS, centrifuge at 300 ⁇ g for 5 min, remove the supernatant, and repeat the above operation once. Add fluorescent secondary antibody PE goat anti-mouse IgGFc, 80 ⁇ L per well, and incubate at 4°C for 30 minutes.
  • VH and VL region cDNAs of hybridoma mouse anti-18F6G3H9, 202E11H10E3 and 211B11H10G5 cloned by PCR were combined with human IgG1 heavy chain constant region (GenBank No. AK303185.1) and kappa light chain constant region (GenBank No. MG815648.1), respectively.
  • the coding DNAs were ligated to construct chimeric heavy and light chains to obtain expression plasmids for the heavy and light chains of the corresponding human-mouse chimeric antibodies c-mAb1, c-mAb2 and c-mAb3.
  • Vectors typically use pcDNA3.1(-) (purchased from Invitrogen) or other eukaryotic expression vectors, replacing the corresponding mouse light and heavy chain constant regions with the constant region sequences of human IgGl heavy chain or kappa light chain.
  • Plasmid extraction was performed using an endotoxin-free mass extraction plasmid kit (Qiagen, Cat. No. 12391), and the specific operation was performed according to the instructions provided by the manufacturer.
  • CHO-S cells were cultured in CD CHO medium (Gibco, Cat. No. 10743-029) according to the manufacturer's instructions in a 37°C, 5% CO2 cell incubator.
  • the plasmids for the light chain sequence were co-transfected into CHO-S cells together, and the two plasmids were co-transfected to express the anti-BCMA monoclonal chimeric antibodies c-mAb1, c-mAb2 and c-mAb3, respectively.
  • the culture temperature was lowered to 32°C, and 3.5% 2 ⁇ EFC+ (Gibco, Cat. No. A2503105) was supplemented daily.
  • the expression supernatant was harvested by centrifugation at 800 ⁇ g. and filtered with a 0.22 ⁇ m filter.
  • the BCMA antibody in the culture supernatant was purified by protein A affinity chromatography and cation exchange chromatography. Purified chimeric antibody concentration was determined by UV absorbance at 280 nm and the corresponding extinction coefficient for each protein. The purity and homogeneity of the antibodies were assessed by SDS-PAGE and SE-HPLC. Or use ion exchange and SEC with Superdex 200 for secondary purification to prepare high-purity antibody samples for later use.
  • ELISA, BiaCore and flow cytometry were used to evaluate the binding activity of human-mouse chimeric mAb to BCMA antigen, and the binding of engineered monkey BCMA-expressing cells HEK293 cynoBCMA to hematoma cell lines U266B1 and NCI-H929.
  • the purpose of the screening assay is to identify antibodies cross-reactive with human BCMA and with cynomolgus BCMA at the molecular and cellular levels.
  • the affinity of the three chimeric antibodies was evaluated by ELISA assay, and the affinity and species crossed antibodies were selected.
  • Test materials BCMA ECD: huBCMA-mFc and cynoBCMA-mFc; PBS buffer; BSA (Bovogen, Catalog No. BSA0.1), TWEEN 20 (Sinopharm, Catalog No. 30189328) and mouse anti-human IgG Fc antibody [HRP] mAb (Mouse Anti -human IgG Fc Antibody [HRP]mAb) (Genscript, Cat. No. A01854), TMB (BD, Cat. No. 55214), H 2 SO 4 (Sinopharm, Cat. No. 10021618).
  • Test method Human, mouse and monkey BCMA antigens were prepared into 0.5 ⁇ g/mL coating solution with PBS buffer respectively, 100 ⁇ L/well was added to the ELISA plate, and after coating overnight at 4°C, the residual liquid of the coating plate was discarded, and 3% BSA was added. , 300 ⁇ L per well, blocked for 3 hours at room temperature. 300 ⁇ L of PBST (PBS containing 0.1% TWEEN20) was added to each well for washing once. The human-mouse chimeric monoclonal antibody, starting at 10 ⁇ g/mL, was diluted 3 times in 9 gradients, and 100 ⁇ L/well was added to the ELISA plate.
  • PBST PBS containing 0.1% TWEEN20
  • the affinity of the three chimeric antibodies to cell surface BCMA antigen was evaluated by FACS method.
  • Test materials cell line NCI-H929, U266B1, HEK293 cynoBCMA, HEK293; PBS buffer; FBS (Fetal Bovine Serum, Gibco, 10099141); PE anti-human IgG Fc (PE anti-human IgG Fc) (Biolegend, 409304).
  • Test method resuspend the cells with PBS, adjust the cell concentration so that the number of cells per well is 2 ⁇ 10 5 .
  • the pre-cooled PBS containing 1% FBS was centrifuged at 300 ⁇ g for 5 min, and the supernatant was removed, and the above operation was repeated once.
  • c-mAb3 also binds to HEK293cynoBCMA expressing monkey BCMA cells with a calculated EC50 value of 2.66 nM.
  • Test materials Biotin labeling kit (DoJindo, LK03); soluble human April (ACRO, Cat. No. APL-H5244); cynoBCMA-his (ACRO, BCA-C52H7) and huBCMA-his (ACRO, BCA-H522y); PBS buffer ; BSA (Bovogen, BSA0.1); TWEEN 20 (Sinopharm, 30189328); Streptavidin-Peroxidase Polymer (Streptavidin-Peroxidase Polymer, Ultrasensitive) (sigma-aldrich, S2438-250UG); TMB Substrate Reagent Set (RUO) (BD, 555214); H 2 SO 4 (Sinopharm, 10021618).
  • BSA Bovogen, BSA0.1
  • TWEEN 20 Seopharm, 30189328
  • Streptavidin-Peroxidase Polymer Streptavidin-Peroxidase Polymer, Ultrasensitive
  • RUO TMB Substrate Rea
  • Test method label soluble human APRIL protein with biotin according to the instructions for use, and label it as APRIL-biotin.
  • 96-well microtiter plates were treated with 100 ⁇ L of 0.5 ⁇ /gmL huBCMA-his prepared in PBS and incubated overnight at 4°C. Plates were then washed three times with 0.1% Tween-20 in PBS wash buffer, followed by the addition of 300 [mu]L of 1% BSA in PBS for 2 hours of blocking.
  • BCMA antibody was added to the plate in a volume of 100 ⁇ L, after 1 hour incubation at 37°C, 80 ng of APRIL-biotin was added to each well and incubated at 37°C for 1 hour.
  • the unbound APRIL-biotin was washed with PBS washing buffer containing 0.1% Tween-20, the secondary antibody streptavidin-peroxidase polymer (sigma-aldrich, S2438-250UG) was added, and the cells were incubated at 37°C for 1 h. Unbound secondary antibody was washed with PBS wash buffer containing 0.1% Tween-20, and TMB chromogenic solution was added at 100 ⁇ L per well. After 5 minutes of reaction at room temperature, 2M H 2 SO 4 was added to stop the reaction, 100 ⁇ L/well. The ELISA plate after the reaction was stopped was placed on a microplate reader (Molecular Devices, SPECTRA Max plus 384), and the absorbance OD450 value was read at a wavelength of 450 nm. The test results are shown in Table 8 and Figure 4.
  • protein A affinity-purified human serum-derived IgG (Sigma I4506) was used as a negative control.
  • the experimental results show that chimeric antibody c-mAb1 and antibody c-mAb2 can inhibit the binding of APRIL to huBCMA-his.
  • c-mAb1 can reach 80% competition
  • c-mAb2 can reach 60%. compete.
  • the affinity of c-mAb3 to huBCMA-his is similar to that of c-mAb1 and c-mAb2, but it failed to effectively inhibit the binding of APRIL to huBCMA-his, indicating that antibody c-mAb3 may have a different binding site than APRIL and huBCMA-his .
  • Example 8 Human-mouse chimeric mAb inhibits the binding of positive antibody 83A10 to BCMA
  • Test materials Biotin labeling kit (DoJindo, LK03); positive antibody 83A10 (patent WO2014122144A1); cynoBCMA-his (ACRO, BCA-C52H7) and huBCMA-his (ACRO, BCA-H522y); PBS buffer; BSA (Bovogen) , BSA0.1); TWEEN 20 (Sinopharm, 30189328); Streptavidin-peroxidase polymer (sigma-aldrich, S2438-250UG); TMB Substrate Reagent Set (RUO) (BD, 555214); H 2 SO 4 (Sinopharm, 10021618).
  • DoJindo LK03
  • positive antibody 83A10 patent WO2014122144A1
  • cynoBCMA-his ACRO, BCA-C52H7
  • huBCMA-his ACRO, BCA-H522y
  • PBS buffer BSA (Bovogen) , BSA
  • Test method label soluble human 83A10 protein with biotin according to the instructions for use (for the sequence, see the clone number 83A10 antibody of US Patent WO2018083204A1, VH corresponds to SEQ ID NO: 19 of the application, VL corresponds to SEQ ID NO: 29 of the application, heavy chain constant region is the heavy chain constant region of human IgG1, and the light chain constant region is the human kappa light chain constant region), labeled as 83A10-biotin.
  • 96-well microtiter plates were treated with 100 ⁇ L of huBCMA-his at 0.5 ⁇ g/mL in PBS and incubated overnight at 4°C.
  • the experimental results show that the antibody c-mAb1 and the antibody c-mAb2 can inhibit the binding of 83A10 to huBCMA-his.
  • c-mAb1 can reach 70% competition, and c-mAb2 can reach 40%. compete.
  • the affinity of c-mAb3 to huBCMA-his is similar to that of c-mAb1 and c-mAb2, but it fails to effectively inhibit the binding of 83A10 to huBCMA-his, indicating that antibody c-mAb3 may have a different binding site than 83A10 and huBCMA-his .
  • c-mAb3 competed 50% for the binding of 83A10 to cynoBCMA-his.
  • the purpose of humanization design is to use the method of 3D modeling to transform the original mouse-derived sequence into human-derived sequence through database alignment to reduce immunogenicity.
  • the main implementation method is to change the murine CDR sequence into a humanized sequence through CDR (complementarity determining region, complementarity determining region) transplantation.
  • the original heavy chain mVH sequence of the c-mAb1 molecule was designed as a humanized sequence huVH1 (SEQ ID NO: 24, VH1);
  • the original heavy chain mVH sequence of the c-mAb2 molecule was designed as a humanized sequence huVH2 (SEQ ID NO: 26, VH2);
  • the original heavy chain mVH sequence of the c-mAb3 molecule was designed as two humanized sequences huVH3 (SEQ ID NO: 28, VH3) and huVH4 (SEQ ID NO: 30, VH4).
  • the original light chain mVL sequence of the c-mAb1 molecule was designed as a humanized sequence huVL1 (SEQ ID NO: 25, VL1);
  • the original light chain mVL sequence of the c-mAb2 molecule was designed as a humanized sequence huVL2 (SEQ ID NO: 27, VL2);
  • the original light chain mVL of the c-mAb3 molecule was designed as two humanized sequences huVL3 (SEQ ID NO: 29, VL3), huVL4 (SEQ ID NO: 31, VL4).
  • c-mAb1 is the parent antibody of hu-mAb1
  • c-mAb2 is the parent antibody of hu-mAb2
  • c-mAb3 is the parent antibody of hu-mAb3 and hu-mAb4.
  • the humanized antibody expression plasmids were respectively expressed in ExpiCHO-S (ATCC, NO.CCL-61) cells, and the humanized antibody protein was obtained after purification. Using ELISA, Biacore and cell affinity detection, four humanized antibodies (designated "hu-mAb1, hu-mAb2, hu-mAb3, hu-mAb4") were obtained.
  • Humanized antibodies were evaluated according to conventional acid stability and thermal stability evaluation methods.
  • the antibody molecule is subjected to protein A affinity chromatography, in the acid elution step (using citrate buffer with pH 3.5), the eluted antibody solution is not neutralized, and after being kept in the buffer for a period of time, 1/10 volume of 1M Tris-HCl (Ph 8.0) was added to neutralize by sampling at 30min, and the sample was detected by HPLC-SEC.
  • the humanized antibody molecules hu-mAb1, hu-mAb2, hu-mAb3 and hu-mAb4 did not aggregate or degrade after being treated at pH 3.5 for 30 min, and the purity was >95%, indicating that they were in an acidic environment. can maintain stability.
  • no aggregation or degradation occurred after being treated at 40°C for 14 days, and the purity was more than 95%, indicating that it could maintain stability at 40°C, as shown in Table 12.
  • the ELISA detection method was used to evaluate the affinity of 4 humanized antibodies, and the humanization evaluation was completed.
  • Test materials BCMAECD: huBCMA-his and cynoBCMA-his; PBS buffer; BSA (Bovogen, BSA0.1), TWEEN 20 (Sinopharm, 30189328); HRP-conjugated 6*His, His-Tag antibody (HRP-Conjugated 6*His, His-Tag Antibody) (Proteintech, HRP-66005-100), TMB (BD, 55214); H 2 SO 4 (Sinopharm, 10021618).
  • Test method huBCMA-his and cynoBCMA-his antigens were prepared with PBS (pH 7.4) into 0.5 ⁇ g/mL coating solution, 100 ⁇ L/well was added to the ELISA plate, and the coating plate residue was discarded after coating overnight at 4°C. Add 3% BSA, 300 ⁇ L per well, and block for 3 hours at room temperature. 300 ⁇ L of PBST was added to each well for washing once, and the humanized single antigen solution was diluted 3-fold in 11 gradients, and 100 ⁇ L/well was added to the ELISA plate.
  • results show that, after humanization transformation, the inventors obtained a humanized monoclonal antibody whose binding activity to huBCMA-his and cynoBCMA-his was comparable to that of the parent antibody within a 3-fold change, and unexpectedly obtained a superior human source with improved affinity. 10%-90% higher affinity than the positive control antibody 83A10. At the same time, the cross-species characteristics are retained, and the binding affinity to monkeys is significantly higher than that of human-mouse chimeric mAbs.
  • antigen-antibody binding kinetics and affinity were determined using the BIACORE method.
  • Test materials human BCMA/TNFRSF17 protein (Human BCMA/TNFRSF17 Protein) (ACRO, BCA-H522y); cynomolgus monkey/rhesus monkey BCMA/TNFRSF17 protein (Cynomolgus/Rhesus macaque BCMA/TNFRSF 17Protein) (ACRO, BCA-C52H7 ); Sereis S Sensor Chip CM5 (GE, BR100530); Anti-histidine antibody (GE, 28995056); HBS-EP (10X) (GE, BR-1006-69); Glycine 10mM, pH 1.5 (GE, BR100354).
  • Test method The anti-histidine antibody (GE, His capture Kit (His capture Kit), product number: 28995056) was coupled to the Sereis S Sensor Chip CM5 chip, the test sample was captured, and the antigen was used as an analyte to detect its relationship with the donor. Kinetic and affinity data for test article binding.
  • the initial concentration of the antigen binding to the test substance is 10nM, on this basis, 2-fold gradient dilution, that is, the concentration of antigen dilution is 10nM, 5nM, 2.5nM, 1.25nM, 0.625nM, and the antigens are sequentially from low concentration to 0.625nM. Start injection at high concentration, and set 1 negative control (i.e.
  • the affinity test results with human/monkey BCMA show that the affinity of the anti-BCMA humanized antibody of the present invention is one to two orders of magnitude higher than that of the control antibody 83A10 (patent WO2014122144A1), and has stronger affinity.
  • Example 13 Humanized antibodies inhibit binding of APRIL ligands to BCMA
  • Test materials Biotin labeling kit (DoJindo, LK03), soluble human APRIL (ACRO, Cat. No. APL-H5244), cynoBCMA-his (ACRO, BCA-C52H7) and huBCMA-his (ACRO, BCA-H522y), TMB Substrate Reagent Set (BD, 555214).
  • Test method the same as in Example 7, the human-mouse chimeric monoclonal antibody was replaced by a humanized monoclonal antibody.
  • the test results are shown in Table 15 and Figure 7.
  • the antibody concentration was 12ug/mL
  • the inhibition rates of hu-mAb1 and hu-mAb2 were 70% and 60%, respectively.
  • hu-mAb3 and hu-mAb4 have high affinity to huBCMA-his, but failed to significantly inhibit the binding of APRIL to huBCMA-his. his binding site.
  • Example 14 Humanized antibody inhibits the binding of positive antibody 83A10
  • test method was the same as that of Example 8, except that the human-mouse chimeric mAb was replaced by a humanized mAb.
  • the affinity of 4 humanized mAbs to cell surface BCMA antigen was evaluated by FACS method.
  • Test material cell line NCI-H929, HEK293 cynoBCMA, HEK293; buffer: 1% FBS-PBS, pH 7.4; PE anti-human IgG Fc (Biolegend, 409304)
  • Test method Preparation of cell suspension: resuspend the cells with PBS, adjust the cell concentration so that the number of cells per well is 2 ⁇ 10 5 . Add 50 ⁇ L of the mouse-derived antibody to be tested to the 96-well plate, the initial concentration of the antibody is 3000nM, 3-fold serial dilution, and incubate at 4°C for 1 hour. After the primary antibody incubation, add 150 ⁇ L of pre-cooled 1% FBS to the 96-well plate. -PBS, centrifuge at 300 ⁇ g for 5 min, remove the supernatant, and repeat the above operation once.
  • Table 17 Comparison of binding activity of humanized antibodies to human/monkey BCMA expressing cell lines
  • Test materials cell lines NCI-H929, U266B1, NK92MI-CD16a (Image Onco Biomedical Technology (Shanghai) Co., Ltd.); PBS buffer; hydroxyfluorescein diacetate succinimidyl ester (5,6-carboxyfluorescein diacetate, succinimidyl ester, CFSE, eBioscience, 65-0850-84); propidium bromide (Propidium iodide, PI, Sigma, P4170).
  • Test method Take target cells NCI-H929, U266B1 and effector cells NK92MI-CD16a for cell counting to detect viability. Take the cells to be used and centrifuge them at 300 ⁇ g for 5 min to collect the cells and stain the cells with 5 ⁇ M CFSE (37°C, 15 min). The medium was washed twice, counted on a VI-Cell cell counter (Beckman), and then added to a 96-well plate according to the experimental design, with 2 ⁇ 10 4 cells/100 ⁇ L per well.
  • the prepared 4 ⁇ antibody molecules were added at 50 ⁇ L/well, and NK92MI-CD16a was added to a 96-well plate (1 ⁇ 10 5 cells/50 ⁇ L per well, and the effect-target ratio was 5:1).
  • the cell culture plate was placed in a cell culture incubator for 6 hours, and then PI was added with a final concentration of 1 ⁇ g/mL. After incubation for 10 minutes, a flow cytometer (BD Accuri TM C6) was used for on-board detection, and CFSE+PI+ double positive cells were analyzed. Percentage of CFSE+ positive cells.

Abstract

一种抗BCMA的抗体或其抗原结合片段，其用于治疗或预防肿瘤中的应用。

Description

抗BCMA抗体及其制备方法和应用 技术领域
本发明涉及医药领域，具体地涉及B细胞成熟抗原(也称B Cell Maturation Antigen，BCMA)的抗体及其制备方法和应用。
背景技术
多发性骨髓瘤又称Multiple Myeloma，简写为MM。MM的发生和发展是一个多步骤的进程，初始为单克隆丙种球蛋白病(monoclonal gammopathy of undetermined significance，MGUS)，逐步进展成为无烟型MM(smoldering MM，SMM)，活跃的MM(active MM)，和浆细胞白血病(plasma cell leukemia，PCL)。据统计，全球发病率11.4万人次/年，呈逐年增长趋势，致死率8万人次/年。高发年龄段在65-74岁，并发症包括高钙血症，肾脏机能不全、贫血和感染。目前，治疗MM的主要方式包括自体干细胞移植，配合传统的化疗药物，免疫调节药物(包括thalidomide，lenalidomide and pomalidomide)，蛋白酶体抑制剂(包括bortezomib，carfilzomib and ixazomib)。虽然初次治疗MM患者对现有疗法均较为敏感，但是MM患者在治疗后期极易出现复发并产生耐药性，因此，对于复发转移的MM患者，新的治疗策略亟待提出，改进治疗现状。
免疫疗法的出现改变了MM的治疗格局。治疗性单抗能够有效的通过其特殊化的Fc与表达Fcγ受体的效应细胞结合，诱导效应细胞产生抗体依赖的细胞毒作用(antibody-dependent cell cytotoxicity，ADCC)，补体依赖的细胞毒作用(complement-dependent cytotoxicity，CDC)以及抗体依赖的细胞介导的胞吞来清除MM细胞。2015年，美国FDA批准2款治疗性单抗靶向CD38的Daratumumab和靶向SLAMF7的Elotuzumab，因其低毒特征，与现有疗法的联合方案应用于新发MM患者和RRMM患者，临床获益明显。但因二者亦表达于正常的活化B淋巴细胞，T淋巴细胞，单个核细胞，自然杀伤细胞(natural killer cells，NK细胞)和其他的效应细胞，在单抗治疗过程中，消除MM细胞的同时会降低相关效应细胞的数量，从而减弱单抗对MM细胞的杀伤效果，同时对正常靶细胞的清除也伴随潜在的非特异杀伤的毒副作用，因此CD38和SLAMF7不是最理想的MM治疗性靶点。
BCMA全称为tumor necrosis factor receptor superfamily member 17，简称TNFRSF17(UniProt Q02223)，又称为B-cell maturation antigen(BCMA)或CD269；在浆母细胞和浆细胞中广泛表达的一类非糖基化的III型跨膜蛋白。相比较于另外两种TNFRs：BAFF-R(B-cell activation factor receptor)和TACI(transmembrane  activator and calcium modulator and cyclophilin ligand interactor)，只有BCMA能参与晚期记忆型B细胞向PC细胞的转化。同时在BCMA敲除的小鼠中发现，BCMA的缺失仅会损伤长期PC(long-term PC)的存活，但不影响早期体液免疫，B细胞发育成熟和短期的免疫球蛋白的分泌。
相较于靶点CD38和SLAMF7，BCMA在MM组织和细胞中特异性高表达，并与MM的疾病进程相关。BAFF-R在MM细胞系或患者MM细胞中几乎无法检出，TACI的阳性比例和表达强度较BCMA弱。在对BCMA的生物学功能的体内外研究中，BCMA的过表达可激活下游AKT，MAPK，NFκB信号通路，诱导关键抗凋亡蛋白Mcl1，Bcl2，Bcl-xL，促微血管新生蛋白CD31和血管内皮生长因子VEGF的表达。BCMA的过表达还可以调节破骨细胞的活化，粘附，血管新生和转移，促进破骨细胞表达或分泌免疫抑制相关蛋白及细胞因子，如PD-L1(programmed death ligand 1)，TGFβ(transforming growth factorβ)和IL-10(interleukin 10)。因此，阻断BCMA可以有效抑制MM细胞的生长，逆转免疫抑制微环境。综上，BCMA是治疗多发性骨髓瘤(MM)的合适的靶点。
发明内容
本发明提供一种针对BCMA的特异性单克隆抗体。
具体地，本发明涉及以下技术方案：
1.抗BCMA抗体或其抗原结合片段，其特征在于包含
(i)SEQ ID NO：1所示重链可变区包含的HCDR1，HCDR2和HCDR3；和/或SEQ ID NO：2所示轻链可变区包含的LCDR1，LCDR2和LCDR3：
(ii)SEQ ID NO：3所示重链可变区包含的HCDR1，HCDR2和HCDR3；和/或SEQ ID NO：4所示轻链可变区包含的LCDR1，LCDR2和LCDR3：
(iii)SEQ ID NO：5所示重链可变区包含的HCDR1，HCDR2和HCDR3；和/或SEQ ID NO：6所示轻链可变区包含的LCDR1，LCDR2和LCDR3：或
(iv)重链可变区包含的HCDR1，HCDR2和HCDR3；和/或轻链可变区包含的LCDR1，LCDR2和LCDR3；其中，所述重链可变区和/或轻链可变区与(i)至(iii)任一所述的重链可变区和/或轻链可变区相比，至少一个CDR含有突变，所述突变为一个或多个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)并能够保留与BCMA结合亲和力；优选地，所述的突变为保守氨基酸突变；
优选地，按照IMGT、Kabat、Chothia或AbM编号***定义；优选地，按照IMGT编号***，所述抗BCMA抗体包含以下CDRs，
(1)HCDR1，其包含SEQ ID NO：7所示的氨基酸序列或其变体，或由其组成，
HCDR2，其包含SEQ ID NO：8所示的氨基酸序列或其变体，或由其组成，
HCDR3，其包含SEQ ID NO：9所示的氨基酸序列或其变体，或由其组成，
LCDR1，其包含SEQ ID NO：16所示的氨基酸序列或其变体，或由其组成，
LCDR2，其包含SEQ ID NO：17所示的氨基酸序列或其变体，或由其组成，和
LCDR3，其包含SEQ ID NO：18所示的氨基酸序列或其变体，或由其组成；
(2)HCDR1，其包含SEQ ID NO：10所示的氨基酸序列或其变体，或由其组成，
HCDR2，其包含SEQ ID NO：11所示的氨基酸序列或其变体，或由其组成，
HCDR3，其包含SEQ ID NO：12所示的氨基酸序列或其变体，或由其组成，
LCDR1，其包含SEQ ID NO：19所示的氨基酸序列或其变体，或由其组成，
LCDR2，其包含SEQ ID NO：20所示的氨基酸序列或其变体，或由其组成，和
LCDR3，其包含SEQ ID NO：21所示的氨基酸序列或其变体，或由其组成；或
(3)HCDR1，其包含SEQ ID NO：13所示的氨基酸序列或其变体，或由其组成，
HCDR2，其包含SEQ ID NO：14所示的氨基酸序列或其变体，或由其组成，
HCDR3，其包含SEQ ID NO：15所示的氨基酸序列或其变体，或由其组成，
LCDR1，其包含SEQ ID NO：22所示的氨基酸序列或其变体，或由其组成，
LCDR2，其包含SEQ ID NO：17所示的氨基酸序列或其变体，或由其组成，和
LCDR3，其包含SEQ ID NO：23所示的氨基酸序列或其变体，或由其组成，
其中，所述变体为与序列编号所示的对应CDR序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性并保留与BCMA结合亲和力的序列，或与序列编号所示的对应CDR序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，***或缺失)并保留与BCMA结合亲和力的氨基酸序列；
优选地，所述抗体或其抗原结合片段的重链可变区和/或轻链可变区包含来自人源、鼠源或兔源的FR区。
2.项目1所述的抗BCMA抗体或其抗原结合片段，其中所述抗体的重链可变区包含下述序列或其变体，或由下述序列或其变体组成：SEQ ID NO：1、 SEQ ID NO：3、SEQ ID NO：5、SEQ ID NO：24、SEQ ID NO：26、SEQ ID NO：28或SEQ ID NO：30；并且
所述抗体的轻链可变区包含下述序列或其变体，或由下述序列或其变体组成：SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：25、SEQ ID NO：27、SEQ ID NO：29或SEQ ID NO：31；
其中所述变体为与序列编号所示的对应的抗体重链可变区或轻链可变区的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、 90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性并保留与BCMA结合亲和力的序列，或与序列编号所示的对应的抗体重链可变区或轻链可变区的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，***或缺失)并保留与BCMA结合亲和力的氨基酸序列。
3.项目1或2所述的抗BCMA抗体或其抗原结合片段，其中
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：1或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：2或其变体所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：3或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：4或其变体所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：5或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：6或其变体所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：24或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：25或其变体所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：26或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：27或其变体所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：28或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：29或其变体所示；或
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：30或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：31或其变体所示。
4.项目1或2所述的抗BCMA抗体或其抗原结合片段，其中所述抗体的重链恒定区和所述抗体的轻链恒定区来自人IgG1，IgG2，IgG3或IgG4，优选为人IgG1重链恒定区，GenBank ACCESSION：AK303185.1；轻链恒定区为Ig1 kappa链恒定区，GenBank ACCESSION：MG815648.1。
5.项目1或2所述的抗BCMA抗体或其抗原结合片段，其中所述抗体为单克隆抗体、部分或全人源化抗体、嵌合抗体、单链抗体、多特异性抗体(例如双特异性抗体)。
6.项目1或2所述的抗BCMA抗体或其抗原结合片段，所述抗原结合片段选自Fab、Fab′、F(ab′) 2、F(ab) 2、Fd、Fv、dAb、Fab/c、互补决定区片段、scFv、scFv多聚体、二硫键稳定性Fv(dsFv)、(dsFv) 2、双特异性dsFv(dsFv-dsFv′)、双链抗体(Diabody)、二硫键稳定的双链抗体(ds-Diabody)、由包含一个或多个CDR的抗体的一部分形成的多特异性抗体、单域抗体(sdab)、纳米抗体、结构域抗体或二价结构域抗体。
7.分离的多肽或其变体，其选自以下各项组成的组：
(1)分离的多肽或其变体，其包含SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的序列；
(2)分离的多肽或其变体，其包含SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的序列；
(3)分离的多肽或其变体，其包含SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：21所示的序列；
(4)分离的多肽或其变体，其包含SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：21所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的序列；
(5)分离的多肽或其变体，其包含SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：22、SEQ ID NO：17和SEQ ID NO：23所示的序列；
(6)分离的多肽或其变体，其包含SEQ ID N0：22、SEQ ID NO：17和SEQ ID NO：23所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还应包含SEQ ID NO：13、SEQ ID NO：14和SEQ ID N0：15所示的序列；
(7)分离的多肽或其变体，其包含SEQ ID NO：1所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：2所示的序列；
(8)分离的多肽或其变体，其包含SEQ ID NO：2所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：1所示的序列；
(9)分离的多肽或其变体，其包含SEQ ID NO：3所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：4所示的序列；
(10)分离的多肽或其变体，其包含SEQ ID NO：4所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：3所示的序列；
(11)分离的多肽或其变体，其包含SEQ ID NO：5所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：6所示的序列；
(12)分离的多肽或其变体，其包含SEQ ID NO：6所示的序列，其中所述多肽 作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：5所示的序列；
(13)分离的多肽或其变体，其包含SEQ ID NO：24所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：25所示的序列；
(14)分离的多肽或其变体，其包含SEQ ID NO：25所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：24所示的序列；
(15)分离的多肽或其变体，其包含SEQ ID NO：26所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：27所示的序列；
(16)分离的多肽或其变体，其包含SEQ ID NO：27所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：26所示的序列；
(17)分离的多肽或其变体，其包含SEQ ID NO：28所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：29所示的序列；
(18)分离的多肽或其变体，其包含SEQ ID NO：29所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：28所示的序列；
(19)分离的多肽或其变体，其包含SEQ ID NO：30所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：31所示的序列；或
(20)分离的多肽或其变体，其包含SEQ ID NO：31所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：30所示的序列，
其中所述变体为与序列编号所示的对应序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性并保留与BCMA结合亲和力的序列，或与序列编号所示的对应序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，***或缺失)并保留与BCMA结合亲和力的氨基酸序列。
8.核酸分子，其编码项目1-6任一项所述的抗体或其抗原结合片段，或项目7所述分离的多肽或其变体。
9.载体，其包含项目8所述的核酸分子。
10.宿主细胞，其包含项目8所述的核酸分子，或项目9所述的载体。
11.偶联物，其包含项目1-6任一项所述的抗体或其抗原结合片段以及偶联部分，其中，所述偶联部分为纯化标签(如His标签)、可检测的标记、药物、毒素、细胞因子、酶、或其组合；优选地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质、化疗剂、生物毒素、聚乙二醇或酶。
12.融合蛋白或多特异性抗体(优选双特异性抗体)，其包含项目1-6任一项所述的抗体或其抗原结合片段；优选地，所述融合蛋白为CAR构建物，所述CAR构建物特异性结合BCMA。
13.试剂盒，其包括项目1-6任一项所述的抗体或其抗原结合片段、项目11所述的偶联物或项目12所述的融合蛋白或多特异性抗体；优选地，所述试剂盒还包括第二抗体，其特异性识别所述抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；优选地，所述试剂盒用于检测BCMA在样品中的存在或其水平。
14.药物组合物，其包含项目1-6任一项所述的抗体或其抗原结合片段、项目11所述的偶联物或项目12所述的融合蛋白或多特异性抗体；可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂；优选地，所述药物组合物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
15.项目1-6任一项所述的抗体或其抗原结合片段、项目11所述的偶联物或项目12所述的融合蛋白或多特异性抗体用于治疗和/或预防肿瘤(例如多发性骨髓瘤)，或在制备治疗和/或预防肿瘤(例如多发性骨髓瘤)的药物中的用途，或者在制备用于诊断肿瘤的药物中的用途。
16.试剂盒，其包含(1)项目1-6任一项所述的抗体或其抗原结合片段、项目11所述的偶联物或项目12所述的融合蛋白或多特异性抗体，和(2)针对其它抗原(如CD38和/或SLAMF7)的抗体或其抗原结合片段，和/或细胞毒性剂，和任选地，使用说明书。
17.治疗或预防肿瘤的(例如多发性骨髓瘤)方法，包括向受试者施用治疗有效量的项目1-6任一项所述的抗体或其抗原结合片段、项目11所述的偶联物或项目12所述的融合蛋白或多特异性抗体。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
在本发明中涉及的术语具备本领域技术人员理解的常规含义。在本技术领域内使用和/或可接受的情况下，一个术语有两个或两个以上定义时，本文使用的术 语的定义用于包括所有的含义。
本领域普通技术人员可以理解，抗体的CDR区负责抗体对抗原的结合特异性。在已知抗体重链和轻链可变区序列的情况下，目前有几种确定抗体CDR区的方法，包括Kabat，IMGT，Chothia和AbM编号***。然而，每种关于抗体或其变体的CDR的定义的应用都将在本文定义和使用的术语的范围内。如果给定该抗体的可变区氨基酸序列，则本领域技术人员通常可确定哪些残基包含特定CDR，而不依赖于该序列自身之外的任何实验数据。
本文使用的，“抗体”或者“抗原结合片段”指特定识别并结合抗原的多肽或多肽复合体。术语“抗体”以广义使用，并且包括免疫球蛋白或抗体分子，所述抗体分子包括单克隆或多克隆的人、人源化、复合和嵌合抗体以及抗体片段。抗体可为整个抗体也可为任何抗体片段、抗原结合片段或者其单链。因此术语“抗体”包括含有特定分子的任何蛋白或肽，该特定分子含有至少一部分的免疫球蛋白分子，该免疫球蛋白分子具有结合至抗原的生物活性。此种情况的实例包括但不限于，重链或轻链或其配体结合部分的互补决定区(CDR)，重链或轻链可变区，重链或轻链恒定区，框架(FR)区或其任何部分，或结合蛋白的至少一部分。在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。本申请的免疫球蛋白分子或抗体分子可为任何类型的(例如IgG、IgE、IgM、IgD、IgA和IgY)、免疫球蛋白分子的任何类(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类。
术语“抗体片段”或“抗原结合片段”包括但不限于，F(ab′) 2、F(ab) 2、Fab′、Fab、Fv、Fd、dAb、Fab/c、互补决定区(CDR)片段、单链Fvs(scFv)、二硫键稳定性Fv(Disulfide-stabilized Fv fragment，dsFv)、(dsFv) 2、双特异性dsFv(dsFv-dsFv′)、双链抗体(Diabody)、二硫键稳定的双链抗体(ds-Diabody)、scFv多聚体(如scFv二聚体、scFv三聚体)、由包含一个或多个CDR的抗体的一部分形成的多特异性抗体、纳米抗体、单域抗体(sdab)、结构域抗体、二价结构域抗体，或任何其他与抗原结合但不包含完整的抗体结构的抗体片段。不管结构如何，抗原结合片段包括任何能够与亲本抗体或亲本抗体片段结合的相同抗原结合的多肽或多肽复合体。Mao C S等“Disulfide stabilized Fv Fragments(dsFv)：a New Type of Engineering Antibody Fragments”.Progress in Biochemistry and Biophysics，1998，25(6)：525-526介绍了dsFv的结构。Holt等″Domain antibodies：proteins for therapy″Trends inBiotechnology(2003)：Vol.21，No.11：484-490综述了称为“结构域抗体”或dAbs的抗原结合片段，它仅含有抗体的VH或VL结构域，因此比例如Fab和scFv更小。dAbs是抗体最小的已知的抗原结合片段，从11kDa到15kDa。术语“抗体片 段”包括适配体、适配体对映体(spiegelmers)和双体(diabodies)。术语“抗体片段”也包括任何合成的或基因改造的蛋白，它们与抗体一样可结合至特定的抗原以形成复合体。通常，抗体片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
本申请的实施方案提供了多种抗BCMA抗体，这些抗体包含至少一个靶向BCMA抗原的抗原结合结构域。结合BCMA抗原的抗原结合结构域为Fab，或ScFv，或重链可变区(VH)-轻链可变区(VL)之间非共价配对(Fv)。任何上述的抗体或多肽还可包括额外的多肽，例如，抗体N端的信号肽，该信号肽用于指导分泌，或如本文所述的其他异源多肽。本发明不仅包括完整的抗体，还包括具有免疫活性的抗体片段或抗体与其他序列形成的融合蛋白。本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链和轻链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链和轻链的可变区相同或至少90％同源性，较佳地至少95％同源性。因此，本发明包括那些具有带CDR的单克隆抗体轻链和重链可变区的分子，只要其CDR与本发明CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明还包括所述抗体的片段、变体、衍生物和类似物。本申请的抗体、抗原结合片段、它们的变体或衍生物，包括但不限于，多克隆抗体、单克隆抗体、多特异性抗体(如双特异性抗体、三特异性抗体等)、人抗体、动物源抗体、人源化的抗体、灵长类化的(primatized)抗体、或嵌合抗体、CDR嫁接和/或修饰的抗体、单链抗体(例如，scFv)、双链抗体、抗原表位结合片段，例如，Fab、Fab′和F(ab′) 2、Fd、Fvs、单链Fvs(scFv)、单链抗体、二硫键连接的Fvs(dsFv)、包含VL结构域或VH结构域的片段、由Fab表达库产生的片段、和抗独特型(idiotypic)(抗-Id)抗体。本发明的抗体片段、抗原结合片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有人BCMA结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些 变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
出于比较两种或更多种氨基酸序列的目的，第一氨基酸序列和第二氨基酸序列之间的“序列同源性”百分数(在本文也称为“氨基酸同源性”)可以通过用[第一氨基酸序列中与第二氨基酸序列中相应位置的氨基酸残基相同的氨基酸残基数目]除以[第一氨基酸序列中的氨基酸残基总数]并且乘以[100％]而计算，其中第二氨基酸序列中氨基酸残基的每一删除、***、替换或添加——与第一氨基酸序列相比——都视作在单一氨基酸残基(位置)上的差异，即，视作本发明所定义的“氨基酸差异”。备选地，两种氨基酸序列之间的序列同一性程度可以使用已知的计算机算法进行计算，诸如NCBI Multiple Alignment(https：//www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi？LINK_LOC＝BlastHomeLink)。例如，在WO 04/037999，EP 0 967 284，EP 1 085 089，WO 00/55318，WO 00/78972，WO 98/49185和GB 2 357 768-A中描述了用于确定序列同一性程度的一些其它的技术、计算机算法和设置。通常，出于按照上文列出的计算方法来确定两种氨基酸序列之间的“序列同一性”的百分数的目的，将具有最大氨基酸残基数目的氨基酸序列视作“第一”氨基酸序列，并且将另一种氨基酸序列视作“第二”氨基酸序列。
此外，在确定两种氨基酸序列之间的序列同一性程度时，专业技术人员可以考虑所谓的“保守”氨基酸替换，其通常可以描述为这样的氨基酸替换，即，其中氨基酸残基被具有相似化学结构的另一种氨基酸残基替换，并且其对所述多肽的功能、活性或其它生物学特性几乎没有或者基本上没有影响。这种保守氨基酸替换是本领域内公知的，例如，从WO 04/037999，GB-A-3357 768，WO 98/49185，WO 00/46383和WO 01/09300中可知；并且可以基于WO 04/037999以及WO 98/49185和其中所引用的其它参考文献的相关教导而选择这种替换的(优选)类型和/或结合。
“保守氨基酸替换”是其中氨基酸残基被具有类似侧链的氨基酸残基替换。具有类似侧链的氨基酸残基家族已在本领域中定义，其包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)，酸性侧链(例如天冬氨酸，谷氨酸)，不带电荷的极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)，非极性侧 链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)，β-支链的侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，免疫球蛋白多肽的非必需氨基酸残基优选被来自相同侧链家族的其他氨基酸残基替换。在另一些实施方案中，一串氨基酸可被结构上类似的氨基酸串替换，后者在顺序上和/或侧链家族的组成上不同。
在下表中提供了保守性氨基酸替换的非限制性实例，其中相似性得分为0或更高表示在这两个氨基酸之间有保守替换。
  C G P S A T D E N Q H K R V M I L F Y W
W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17
Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10  
F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9    
L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6      
I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5        
M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6          
V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4            
R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6              
K -5 -2 -1 0 -1 0 0 0 1 1 0 5                
H -3 -2 0 -1 -1 -1 1 1 2 3 6                  
Q -5 -1 0 -1 0 -1 2 2 1 4                    
N -4 0 -1 1 0 0 2 1 2                      
E -5 0 -1 0 0 0 3 4                        
D -5 1 -1 0 0 0 4                          
T -2 0 0 1 1 3                            
A -2 1 1 1 2                              
S 0 1 1 1                                
P -3 -1 6                                  
G -3 5                                    
C 12                                      
在一些实施方案中，所述保守替换优选地是这样的替换，即，其中下列组(a)-(e)内的一个氨基酸被同组内的另一氨基酸残基替换：(a)小的脂肪族、非极性或弱极性的残基：Ala，Ser，Thr，Pro和Gly；(b)极性、带负电荷的残基及其(不带电荷的)酰胺：Asp，Asn，Glu和Gln；(c)极性、带正电荷的残基：His，Arg和Lys；(d)大的脂肪族、非极性残基：Met，Leu，Ile，Val和Cys；以及(e)芳族残基：Phe，Tyr和Trp。
特别优选的保守替换如下：Ala替换成Gly或替换成Ser；Arg替换成Lys；Asn替换成Gln或替换成His；Asp替换成Glu；Cys替换成Ser；Gln替换成Asn；Glu替换成Asp；Gly替换成Ala或替换成Pro；His替换成Asn或替换成Gln；Ile替换成Leu或替换成Val；Leu替换成Ile或替换成Val；Lys替换成Arg，替换成Gln或替换成Glu；Met替换成Leu，替换成Tyr或替换成Ile；Phe替换成Met，替换成Leu或替换成Tyr；Ser替换成Thr；Thr替换成Ser；Trp替换成Tyr；Tyr替换成Trp；和/或Phe替换成Val，替换成Ile或替换成Leu。
在一些实施方案中，本发明所述抗体可以结合治疗剂、药物前体、肽、蛋白、酶、病毒、脂质、生物反应调节剂、药剂或PEG。本发明抗体可以连接至或融合至治疗剂上，该治疗剂可包括可检测标记物，如放射性标记物、免疫调节剂、激素、酶、寡核苷酸、光活性治疗剂或诊断剂、细胞毒性剂，其可为药物或毒素，超声增强剂，非放射性标记物，它们的组合和其他这类本领域已知的成分。
在一些实施方案中，本发明所述的抗BCMA抗体例如具有如下一种或多种优点：
(a)本发明抗体具有优异的生物活性和特异性，并与BCMA具有很高的亲和力，比现有BCMA抗体(如83A10抗体)的结合活性更高，并且无明显潜在毒副作用。
(b)本发明的抗体经过人源化改造，仍然保持高亲和力，且降低了免疫原性。
(c)本发明抗体与食蟹猴的BCMA有结合活性，便于在动物模型中进行测试和进行质控检测。
(d)本发明抗体稳定性好，尤其在酸性环境和热处理条件下仍保持良好稳定性。
附图说明
图1.显示了构建稳定转染细胞株HEK293 huBCMA，HEK293 cynoBCMA以及HEK293 mBCMA，所对应的人(A)，猴(B)，鼠(C)全长BCMA的表达质粒图谱。
图2.显示了FACS法对人，猴，鼠BCMA稳定转染细胞株HEK293 huBCMA，HEK293 cynoBCM以及HEK293 mBCMA的BCMA表达鉴定。
图3.显示了FACS法检测18F6G3H9，202E11H10E3和211B11H10G5三份杂交瘤上清中抗体的人猴交叉性检测。
图4.显示了竞争ELISA检测嵌合抗体c-mAb1，c-mAb2，c-mAb3对天然配体APRIL与人BCMA结合的抑制情况。
图5.显示了竞争ELISA检测嵌合抗体c-mAb1，c-mAb2，c-mAb3对阳性对照抗体83A10与人BCMA结合的抑制情况。
图6.显示了竞争ELISA检测嵌合抗体c-mAb3对阳性对照抗体83A10与猴BCMA结合的抑制情况。
图7.显示了竞争ELISA检测人源化抗体hu-mAb1，hu-mAb2，hu-mAb3，hu-mAb4对天然配体APRIL与人BCMA结合的抑制情况。
图8.显示了竞争ELISA检测人源化抗体hu-mAb1，hu-mAb2，hu-mAb3，hu-mAb4对阳性对照抗体83A10与人BCMA结合的抑制情况。
图9.显示了竞争ELISA检测人源化抗体hu-mAb3，hu-mAb4对阳性对照抗体83A10与猴BCMA结合的抑制情况。
图10.显示了人源化单抗hu-mAb1，hu-mAb2，hu-mAb3，hu-mAb4介导效应细胞NK92MI-CD16a对靶细胞NCI-H929的ADCC作用。
图11.显示了人源化单抗hu-mAb1，hu-mAb2，hu-mAb3，hu-mAb4介导效应细胞NK92MI-CD16a对靶细胞U266B1的ADCC作用。
具体实施方式
下面通过实施例详细描述本发明。本领域的普通技术人员可以理解，下述实施例仅是用于举例说明的目的。本发明的精神和范围由权利要求所限定。下述实施例中所用方法如无特别说明均为常规方法，所用的试剂如无特别说明均为可商购的试剂。
实施例1：材料制备
1.1 BCMAECD分子
对应人BCMA的第1至54位氨基酸(SEQ ID NO：32)的重组人BCMA-huFc和BCMA-mFc融合蛋白，后简称huBCMA-huFc和huBCMA-mFc；对应食蟹猴BCMA的第1至53位氨基酸(SEQ ID NO：33)的重组食蟹猴BCMA-huFc和BCMA-mFc，后简称cynoBCMA-huFc和cynoBCMA-mFc；对应小鼠BCMA的第1至49位氨基酸(SEQ ID NO：34)的重组鼠BCMA-huFc和BCMA-mFc融合蛋白，后简称mBCMA-huFc和mBCMA-mFc。以上序列信息来自于美国国家生物技术信息中心。人BCMA的重组人hu BCMA-his融合蛋白(ACR0Biosystem，货号为BCA-H522y)和猴BCMA的重组食蟹猴cynoBCMA-his，(ACR0Biosystem，货号为BCA-C52H7)应用于表征分析。这些材料用于结合和亲和力测量。BCMA ECD分子的氨基酸序列信息如表1。
表1：BCMAECD序列信息
Figure PCTCN2020136748-appb-000001
1.2 BCMA细胞系的建立
使用标准方法将呈递人BCMA(图1A)，食蟹猴BCMA(图1B)和小鼠BCMA(图1C)的载体瞬时转染到HEK293细胞中。通过FACS分选出绿色荧光蛋白阳性的单克隆进行培养，后使用抗人BCMA抗体(Biolegend，货号357504)，抗 食蟹猴BCMA抗体83A10(序列参见美国专利WO2018083204A1的克隆号83A10抗体，VH对应该申请的SEQ ID NO：19，VL对应该申请的SEQ ID NO：29，GenBank No.AK303185.1的人IgG1的重链恒定区和GenBank No.MG815648.1的人K轻链恒定区)和抗鼠BCMA抗体(R&D System，货号MAB593)检测细胞表面受体表达，建立HEK293稳定表达的细胞株(图2)，后称HEK293huBCMA，HEK293cynoBCMA以及HEK293 mBCMA。
从图2可以看出，相对于阴性对照细胞HEK293，稳定转染细胞株HEK293 huBCMA，HEK293 cynoBCMA和HEK293 mBCMA的偏移率分别为93.5％，92.4％和98.6％，表明HEK293 huBCMA，HEK293 cynoBCMA以及HEK293 mBCMA细胞株在细胞表面稳定表达BCMA。
实施例2：BCMA单克隆抗体表达型杂交瘤的制备与分离
免疫方案如下进行：用huBCMA-his融合体作为抗原，与佐剂乳化后对BALB/c，C57BL/6，SJL，ICR小鼠和SD大鼠(购自南模生物)进行多点皮下/腹腔免疫，监测免疫小鼠/大鼠血清效价，若未达效价进行多次免疫。达到要求后取动物的脾细胞与骨髓瘤(Sp2/0)细胞进行电融合，经过HAT筛选，获得杂交瘤多克隆细胞，采用ELISA和FACS的方式筛选阳性多克隆，后用有限稀释的方法将阳性克隆进行亚克隆，以获得稳定单一的阳性杂交瘤细胞，采用ELISA和FACS的方式进行阳性克隆筛选。经筛选得到三株与BCMA结合活性非常高的单克隆细胞株18F6G3H9、202E11H10E3和211B11H10G5，如表2和图3所示。对这三株单克隆细胞株的杂交瘤上清液进行测序、克隆和表达，其中测序结果如实施例3所示，以进一步构建为人IgG1嵌合抗体和完成单抗人源化。
实施例3：抗BCMA抗体V-基因序列克隆测序
Trizol法提取RNA，反转录后获得cDNA；扩增并获得重链和轻链可变区；将PCR产物进行文库构建并进行质量控制；使用Miseq 2 X 300 PE进行高通量测序；生物信息学分析，比对测序结果到IMGT数据库，确定CDR序列。3株鼠源BCMA抗体的V区测序结果如下，CDR序列见表3：
(1)克隆18F6G3H9：
BCMA-HC1 SEQ ID NO：1
EVQLQQSGPELVKPGTSVEMSCKAS GYTFTDYNIHWVRQRHGESLEWLGY INPNNGVTNFNQKFKGKATLTVNKSSSTAYMELRSLT SDDSAVYY CSSGLFD GYPFPYWGQGTLVTVSA；其中，下划线为CDR1、CDR2、CDR3(SEQ ID NO：7、8和9)。
BCMA-LC1 SEQ ID NO：2
DIVLTQSPASLVVSLGQRATISCRAS KSVSTSGYNYIHWYQQKPGQPPKLLIF LASNLKSGVPARFSGGGSGTDFTLNIHPVEEDDAATYF CQHNRELPYTFGGGTKVEIK；其中，下划线为CDR1、CDR2、CDR3(SEQ ID NO：16、17和18)。
(2)克隆202E11H10E3：
BCMA-HC2 SEQ ID NO：3：
QVQLQQSDAELVKPGTSVKISCKVS GYTFTDHTIHWMKQRPEQGLEWIGY IYPRNGNTKYNEKFKGKATLTADKSSSTAYMHLNSLTSEDSAVYF CARYDYD GYFDVWGTGTTVTVSS；其中，下划线为CDR1、CDR2、CDR3(SEQ ID NO：10、11和12)。
BCMA-LC2 SEQ ID NO：4：
DVVMTQTPLSLPVSLGDQASISCRSS QSHVHSDGHTYLHWYLQKPGQSPELLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYF CSQNTHFPWTFGGGTKLEIK，其中，下划线为CDR1、CDR2、CDR3(SEQ ID NO：19、20和21)。
(3)克隆211B11H10G5：
BCMA-HC3 SEQ ID NO：5：
EVQLQQSGPELVKPGASVKILCKAS GYTITDYNMDWVKRSHGKSLEWIGN INPHNGGSIYNQKFKGKATLTVDKSSSTAFMELRSLTSEDTAVYY CARGDAT LVLDYWGQGTSLTVSS；其中，下划线为CDR1、CDR2、CDR3(SEQ ID NO：13、14和15)。
BCMA-LC3 SEQ ID NO：6：
DIVLTQSPASLAVSLGQRATISCRAS KSVSTSGLSYMHWYQQKPGQPPKVLIY LASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYY CQHSRELPPTFGAGTKLELK；其中，下划线为CDR1、CDR2、CDR3(SEQ ID NO：22、17和23)。
Figure PCTCN2020136748-appb-000002
实施例4：结合至BCMA的鼠源BCMA抗体细胞的表征
通过蛋白A亲和层析纯化得到小鼠腹水中的BCMA抗体。通过在280nm处的UV吸光度以及每种蛋白对应的消光系数测定纯化的小鼠BCMA抗体浓度。通过SDS-PAGE评估抗体的纯度(纯度均＞90％)，并使用LAL assay确定抗体的内毒素含量(内毒素含量均＜3EU/mg)。纯化杂交瘤上清获得的鼠源单抗18F6G3H9、202E11H10E3和211B11H10G5，使用ELISA，BiaCore分子结合测定和流式细胞术评估这3个鼠源BCMA抗体与BCMA抗原，经改造的BCMA表达型细胞HEK293 cynoBCMA和癌细胞系U266B1、NCI-H929的结合，其中U266B1、NCI-H929均购自中国典型物培养中心。筛选测定的目的是在分子及细胞层面，鉴定鼠源抗体与人BCMA以及与食蟹猴BCMA交叉反应性。
4.1使用间接ELISA方法测定鼠源单抗
使用ELISA检测方法评估3株鼠源抗体与BCMA抗原的亲和力，确定亲和力及种属交叉性。
试验材料：BCMAECD：huBCMA-huFc和cynoBCMA-huFc；PBS缓冲液(磷酸盐缓冲液(Phosphate Buffered Saline)，pH 74，Gibco，C10010500BT)；BSA(Bovogen，BSA0.1)，TWEEN 20(国药，30189328)和HRP羊抗鼠IgG(HRP Goat Anti-Mouse IgG)抗体(Abclonal，AS003)，TMB(BD，55214)，H 2SO 4(国药，10021618)。
试验方法：将人、鼠、猴的BCMA抗原分别用PBS缓冲液配制成0.5μg/mL包被液，100μL/孔加入酶标板，4℃包被过夜后弃包板残液，加入3％BSA，每孔300μL，室温封闭3小时。每孔加入300μL的PBST(含0.1％TWEEN20的PBS)洗1次，将三种鼠源BCMA抗体，以10μg/mL为起始浓度，按3倍稀释7个梯度，100μL/孔加入酶标板。室温孵育1小时，每孔加300μL的PBST，洗涤3次后加入1％BSA-PBST稀释25000倍的HRP羊抗鼠IgG抗体，100μL/孔加样。室温孵育1小时后，每孔加300μL的PBST，洗涤3次，拍干。加入TMB显色液，每孔100μL。室温反应5分钟后加2M H 2SO 4终止反应，100μL/孔。将中止反应的酶标板置酶标仪(Molecular Devices，SPECTRA Max plus 384)上，读取450nm波长读取吸光度OD450值。试验结果如表4所示。
表4：鼠源抗体对不同种属BCMA结合活性比较
Figure PCTCN2020136748-appb-000003
结果表明，18F6G3H9、202E11H10E3和211B11H10G5鼠源抗体均能与人的BCMA结合，并且均具有很高的结合活性，EC 50值均低于80pM。其中 211B11H10G5还可以与猴的BCMA结合。
4.2使用FACS方法测定鼠源单抗
使用FACS方法评估3株鼠源抗体与细胞表面BCMA抗原的亲和力。
试验材料：细胞株NCI-H929，U266B1，HEK293 cynoBCMA，HEK293；PBS缓冲液；FBS(Fetal Bovine Serum，Gibco，10099141)；PE羊抗鼠IgG Fc(PE Goat anti-mouse IgG Fc)(Biolegend，405307)。
试验方法：用PBS重悬细胞，制备细胞悬液，使每孔细胞数量为200000。在96孔板中加入待检鼠源BCMA抗体50μL，抗体起始浓度3000nM，3倍梯度稀释，4℃孵育1小时，一抗孵育结束后，向96孔板中加入150μL预冷的含1％FBS的PBS，300×g离心5min后去上清，重复上述操作1次。加入荧光二抗PE羊抗鼠IgGFc，每孔80μL，4℃孵育30分钟，二抗孵育结束后，向96孔板中加入150μL预冷的含1％FBS的PBS，用枪头吹打混匀，将96孔板放入板式离心机中配平，300×g离心5min后去上清，重复上述操作1次。100μL含1％FBS的PBS重悬，流式细胞仪(BD Accuri TM C6)检测。试验结果如表5所示。
表5：鼠源抗体与人或猴BCMA表达细胞株的结合活性比较
样品 U266B1，EC50 NCI-H929，EC50 HEK293 cynoBCMA，EC50
18F6G3H9 1.6nM 3.1nM -
202E11H10E3 1nM 1.1nM -
211B11H10G5 24.5nM 11.9nM 30.89nM
结果表明，18F6G3H9、202E11H10E3和211B11H10G5鼠源抗体均能与高表达或中低表达人BCMA的细胞NCI-H929和U266B1细胞结合，单抗亲和力均在50nM以内；同时211B11H10G5可以与猴BCMA结合。亲和力与该抗体同人BCMA亲和力相当。
实施例5：BCMA嵌合抗体表达与纯化
5.1嵌合抗体的质粒制备
通过将PCR克隆的杂交瘤鼠抗18F6G3H9，202E11H10E3和211B11H10G5的VH和VL区cDNA分别与人IgG1重链恒定区(GenBank No.AK303185.1)和kappa轻链恒定区(GenBank No.MG815648.1)的编码DNA连接，来构建嵌合重链和轻链，以获得相应人鼠嵌合抗体c-mAb1，c-mAb2和c-mAb3的重链和轻链的表达质粒。载体一般用pcDNA3.1(-)(购自Invitrogen)或其他真核表达载体，用人IgG1重链或kappa轻链的恒定区序列替换相应的小鼠轻重链恒定区。
5.2嵌合抗体的表达与纯化
利用无内毒素大量提取质粒试剂盒(Qiagen，货号12391)进行质粒提取，具体操作按照厂商提供的说明书进行。CHO-S细胞培养根据厂商提供的说明书在CD CHO培养基(Gibco，货号10743-029)中，置于37℃，5％CO 2细胞培养箱中进行培养，准备好细胞后，将含有重/轻链序列的质粒一起共转染到CHO-S细胞中，共转染这两种质粒以分别表达抗BCMA的单克隆嵌合抗体c-mAb1、c-mAb2和c-mAb3。在转染后的第二天，培养温度下调到32℃，并每天补加3.5％2×EFC+(Gibco，货号A2503105)，培养14天后，800×g离心收获表达上清。并用0.22μm滤膜过滤。通过蛋白A亲和层析和阳离子交换层析纯化得到培养上清液中的BCMA抗体。通过在280nm处的UV吸光度以及每种蛋白对应的消光系数测定纯化的嵌合抗体浓度。通过SDS-PAGE和SE-HPLC评估抗体的纯度和均质性。或者使用离子交换和Superdex 200的SEC进行二次纯化制备高纯度抗体样品备用。
实施例6：BCMA嵌合抗体与BCMA的结合活性
使用ELISA，BiaCore和流式细胞术等方法评估人鼠嵌合单抗与BCMA抗原的结合活性，经改造的猴BCMA表达型细胞HEK293 cynoBCMA和血液瘤细胞系U266B1、NCI-H929的结合。筛选测定的目的是在分子及细胞层面，鉴定抗体与人BCMA以及与食蟹猴BCMA交叉反应性。
6.1间接ELISA——嵌合单抗的测定方法
使用ELISA检测方法评估3株嵌合抗体亲和力，选定亲和力及种属交叉抗体。
试验材料：BCMA ECD：huBCMA-mFc和cynoBCMA-mFc；PBS缓冲液；BSA(Bovogen，货号BSA0.1)，TWEEN 20(国药，货号30189328)和鼠抗人IgG Fc抗体[HRP]mAb(Mouse Anti-human IgG Fc Antibody[HRP]mAb)(Genscript，货号A01854)，TMB(BD，货号55214)，H 2SO 4(国药，货号10021618)。
试验方法：分别将人、鼠、猴BCMA抗原用PBS缓冲液配制成0.5μg/mL包被液，100μL/孔加入酶标板，4℃包被过夜后弃包板残液，加入3％BSA，每孔300μL，室温封闭3小时。每孔加入300μL的PBST(含0.1％TWEEN20的PBS)洗1次，将人鼠嵌合单抗，以10μg/mL起始，按3倍稀释9个梯度，100μL/孔加入酶标板。室温孵育1小时，每孔加300μL的PBST，洗涤3次后加入1％BSA-PBST稀释25000倍的鼠抗人IgG Fc抗体[HRP]mAb抗体，100μL/孔加样。室温孵育1小时后，每孔加300μL的PBST，洗涤3次，拍干。加入TMB显色液，每孔100μL。室温反应5分钟后加2MH 2SO 4终止反应，100μL/孔。将中止反应的酶标板置酶标仪(Molecular Devices，SPECTRAMax plus 384)上，读取450nm波长读取吸光度OD450值。试验结果见表6。
表6：嵌合抗体对不同种属BCMA结合活性比较
Figure PCTCN2020136748-appb-000004
结果证明，嵌合抗体c-mAb1、c-mAb2和c-mAb3均能与huBCMA-mFc结合，EC50值均小于1000pM，其中c-mAb3可以与cynoBCMA-mFc结合，且计算的EC50值为722.5pM。
6.2 FACS-嵌合单抗的筛选方法
使用FACS方法评估3株嵌合抗体与细胞表面BCMA抗原的亲和力。
试验材料：细胞株NCI-H929，U266B1，HEK293 cynoBCMA，HEK293；PBS缓冲液；FBS(Fetal Bovine Serum，Gibco，10099141)；PE抗人IgG Fc(PE anti-human IgG Fc)(Biolegend，409304)。
试验方法：用PBS重悬细胞，调整细胞浓度，使每孔细胞数量为2×10 5。在96孔板中加入待检人鼠嵌合单抗50μl，抗体起始浓度3000nM，3倍梯度稀释，9个梯度，4℃孵育1小时，一抗孵育结束后，向96孔板中加入150μL预冷的含1％FBS的PBS，300×g离心5min后去上清，重复上述操作1次。加入荧光二抗PE抗人IgG Fc(Biolegend，409304)，每孔80μL，4℃孵育30分钟，二抗孵育结束后，向96孔板中加入150μL预冷的含1％FBS的PBS，300×g离心5min后用去上清，重复上述操作1次。100μL含1％FBS的PBS重悬，流式细胞仪(BD Accuri TM C6)检测。试验结果如表7所示。
表7：嵌合抗体与人或猴BCMA表达细胞株的结合活性比较
Figure PCTCN2020136748-appb-000005
结果证明，嵌合抗体均能与高表达人BCMA细胞(NCI-H929)或中低表达BCMA的细胞(U266B1)结合。c-mAb3还可以与表达猴BCMA细胞HEK293cynoBCMA结合，且计算的EC50值为2.66nM。
实施例7：BCMA嵌合抗体抑制APRIL配体与BCMA的结合
利用APRIL结合竞争ELISA实验，评价抗-BCMA嵌合抗体阻断April与BCMA抗原结合的能力。
试验材料：Biotin标记试剂盒(DoJindo，LK03)；可溶性人April(ACRO，货号APL-H5244)；cynoBCMA-his(ACRO，BCA-C52H7)和huBCMA-his(ACRO，BCA-H522y)；PBS缓冲液；BSA(Bovogen，BSA0.1)；TWEEN 20(国药，30189328)；链霉亲和素-过氧化物酶聚合物(Streptavidin-Peroxidase Polymer，Ultrasensitive)(sigma-aldrich，S2438-250UG)；TMB Substrate Reagent Set(RUO)(BD，555214)；H 2SO 4(国药，10021618)。
试验方法：按照使用说明biotin标记可溶性人APRIL蛋白，标记为 APRIL-biotin。用100μL的0.5μ/gmL在PBS中制备的huBCMA-his处理96孔酶标板，并在4℃孵育过夜。然后将板用含有0.1％Tween-20的PBS洗涤缓冲液洗涤三次，接着加入300μL含有1％BSA的PBS封闭2小时。将BCMA抗体以100μL体积加入板中，37℃孵育1小时后，每孔加入80ng的APRIL-biotin，并将在37℃孵育1h。用含有0.1％Tween-20的PBS洗涤缓冲液洗涤未结合的APRIL-biotin，加入二抗链霉亲和素-过氧化物酶聚合物(sigma-aldrich，S2438-250UG)，37℃孵育1h，用含有0.1％Tween-20的PBS洗涤缓冲液洗涤未结合的二抗，加入TMB显色液，每孔100μL。室温反应5分钟后加2M H 2SO 4终止反应，100μL/孔。将中止反应的酶标板置酶标仪(Molecular Devices，SPECTRA Max plus 384)上，读取450nm波长读取吸光度OD450值。试验结果见表8和图4。
表8：嵌合抗体竞争抑制APRIL配体结合人BCMA活性比较
样品 嵌合抗体对应克隆 IC50(ng/mL)
APRIL - 24.67
c-mAb1 18F6G3H9 11.37
c-mAb2 202E11H10E3 10.77
c-mAb3 211B11H10G5 2.748
本实验选用经protein A亲和纯化的人血清来源的IgG(hIgG)(Sigma I4506)作为阴性对照。实验结果证明，嵌合抗体c-mAb1和抗体c-mAb2能够抑制APRIL与huBCMA-his的结合，当抗体浓度到达12ug时，c-mAb1可达80％的竞争，c-mAb2可达60％的竞争。c-mAb3与huBCMA-his的亲和力与c-mAb1，c-mAb2相近，但未能有效抑制APRIL与huBCMA-his的结合，表明抗体c-mAb3可能具备不同于APRIL与huBCMA-his的结合位点。
实施例8：人鼠嵌合单抗抑制阳性抗体83A10与BCMA的结合
评价抗BCMA抗体阻断阳性对照抗体83A10(专利WO2014122144A1)与BCMA抗原结合的能力。
试验材料：Biotin标记试剂盒(DoJindo，LK03)；阳性抗体83A10(专利WO2014122144A1)；cynoBCMA-his(ACRO，BCA-C52H7)和huBCMA-his(ACRO，BCA-H522y)；PBS缓冲液；BSA(Bovogen，BSA0.1)；TWEEN 20(国药，30189328)；链霉亲和素-过氧化物酶聚合物(sigma-aldrich，S2438-250UG)；TMB Substrate Reagent Set(RUO)(BD，555214)；H 2SO 4(国药，10021618)。
试验方法：按照使用说明biotin标记可溶性人83A10蛋白(序列参见美国专利WO2018083204A1的克隆号83A10抗体，VH对应该申请的SEQ ID NO：19，VL对应该申请的SEQ ID NO：29，重链恒定区为人IgG1的重链恒定区，轻链恒定区为人k轻链恒定区)，标记为83A10-biotin。用100μL的0.5μg/mL在PBS中制 备的huBCMA-his处理96孔酶标板，并在4℃孵育过夜。然后将板用含有0.1％Tween-20的PBS洗涤缓冲液洗涤三次，接着加入300μL含有1％BSA的PBS封闭2小时。将BCMA抗体以100μL体积加入板中，37℃孵育1小时后，每孔加入5ng/ml的83A10-biotin，并将板在37℃孵育1h。用含有0.1％Tween-20的PBS洗涤缓冲液洗涤未结合的83A10-biotin，并使用二抗链霉亲和素-过氧化物酶聚合物，37℃孵育1h，用含有0.1％Tween-20的PBS洗涤缓冲液洗涤未结合的二抗，加入TMB显色液，每孔100μL。室温反应5分钟后加2M H 2SO 4终止反应，100μL/孔。将中止反应的酶标板置酶标仪(Molecular Devices，SPECTRA Max plus 384)上，读取450nm波长读取吸光度OD450值。试验结果如表9和图5、图6所示。
表9：嵌合抗体竞争抑制抗体83A10结合人BCMA活性比较
Figure PCTCN2020136748-appb-000006
实验结果证明，抗体c-mAb1和抗体c-mAb2能够抑制83A10与huBCMA-his的结合，当抗体浓度到达7μg/mL时，c-mAb1可达70％的竞争，c-mAb2可达40％的竞争。c-mAb3与huBCMA-his的亲和力与c-mAb1，c-mAb2相近，但未能有效抑制83A10与huBCMA-his的结合，表明抗体c-mAb3可能具备不同于83A10与huBCMA-his的结合位点。c-mAb3在7ug/mL时，可达50％的竞争83A10与cynoBCMA-his的结合。
实施例9：人源化单抗构建制备
人源化设计的目的是利用3D建模的方法，经数据库比对，将原始鼠源序列改造成人源序列以降低免疫原性。主要实施方法是通过CDR(complementarity determining region，互补决定区)移植，将鼠源CDR序列改变成人源化序列。
重链设计结果：
c-mAb1分子原始重链mVH序列设计为1个人源化序列huVH1(SEQ ID NO：24，VH1)；
c-mAb2分子原始重链mVH序列设计为1个人源化序列huVH2(SEQ ID NO：26，VH2)；
c-mAb3分子原始重链mVH序列设计为2个人源化序列huVH3(SEQ ID NO：28，VH3)、huVH4(SEQ ID NO：30，VH4)。
轻链设计结果：
c-mAb1分子原始轻链mVL序列设计为1个人源化序列huVL1(SEQ ID NO：25，VL1)；
c-mAb2分子原始轻链mVL序列设计为1个人源化序列huVL2(SEQ ID NO：27，VL2)；
c-mAb3分子原始轻链mVL设计为2个人源化序列huVL3(SEQ ID NO：29，VL3)、huVL4(SEQ ID NO：31，VL4)。
将以上设计的8条序列组合为4对人源化抗体hu-mAb1(VH1+VL1)、hu-mAb2(VH2+VL2)、hu-mAb3(VH3+VL3)、hu-mAb4(VH4+VL4)进行后续表达验证。其中c-mAb1为hu-mAb1的母抗体，c-mAb2为hu-mAb2的母抗体，c-mAb3为hu-mAb3和hu-mAb4的母抗体。
具体序列信息如下所示：
表10：人源化单抗序列信息
Figure PCTCN2020136748-appb-000007
将人源化抗体表达质粒分别经ExpiCHO-S(ATCC，NO.CCL-61)细胞分别表达，纯化后得到人源化抗体蛋白。利用ELISA，Biacore和细胞亲和力检测，获得了4种人源化抗体(命名为“hu-mAb1，hu-mAb2，hu-mAb3，hu-mAb4”)。
实验结果表明，与鼠源抗体相比，人源化抗体具有相近或更好的亲和力和特异性，见实施例10。
实施例10：人源化抗体的酸稳定性和热稳定性评估
按照常规酸稳定性和热稳定性评估方法对人源化抗体进行评估。抗体分子进行protein A亲和层析时，在酸洗脱步骤中(使用pH3.5的柠檬酸缓冲液)，洗脱下来的抗体溶液不进行中和，在该缓冲液中保持一段时间后，在第30min取样加入1/10体积的1M Tris-HCl(Ph 8.0)进行中和，并进行该样品的HPLC-SEC检测。如表11所示，人源化抗体分子hu-mAb1，hu-mAb2，hu-mAb3和hu-mAb4在经过pH 3.5处理30min后未出现聚集或降解现象，纯度＞95％，说明其在酸性环境中能保持稳定性。同时，在经40℃处理14天后未出现聚集或降解现象，纯度＞95％，说明其在40℃环境中能保持稳定性，如表12所示。
表11 人源化抗体酸稳定性评估结果
Figure PCTCN2020136748-appb-000008
表12 人源化抗体热稳定性评估结果
Figure PCTCN2020136748-appb-000009
实施例11 Elisa法测定人源化抗体分子亲和力
本实施例使用ELISA检测方法评估4个人源化抗体亲和力，完成人源化评估。
试验材料：BCMAECD：huBCMA-his和cynoBCMA-his；PBS缓冲液；BSA(Bovogen，BSA0.1)，TWEEN 20(国药，30189328)；HRP-偶联6*His，His-Tag抗体(HRP-Conjugated 6*His，His-Tag Antibody)(Proteintech，HRP-66005-100)，TMB(BD，55214)；H 2SO 4(国药，10021618)。
试验方法：将huBCMA-his和cynoBCMA-his抗原用PBS(pH7.4)配制成0.5μg/mL包被液，100μL/孔加入酶标板，4℃包被过夜后弃去包板残液，加入3％BSA，每孔300μL，室温封闭3小时。每孔加入300μL的PBST洗1次，将人源化单抗原液，按3倍稀释11个梯度，100μL/孔加入酶标板。室温孵育1小时， 每孔加300μL的PBST，洗涤3次后加入1％BSA-PBST稀释25000倍的HRP-偶联6*His，His-Tag抗体，100μL/孔加样。室温孵育1小时后，每孔加300μL的PBST，洗涤3次，拍干。加入TMB显色液，每孔100μL。室温反应5分钟后加2M H 2SO 4终止反应，100μL/孔。将中止反应的酶标板置酶标仪(Molecular Devices，SPECTRA Max plus 384)上，读取450nm波长读取吸光度OD450值。试验结果见表13。
表13：人源化抗体对人BCMA及猴BCMA结合活性比较
Figure PCTCN2020136748-appb-000010
结果证明，经过人源化改造，本发明人获得了对huBCMA-his和cynoBCMA-his结合活性与母抗体相当的人源化单抗亲和力差异在3倍变化以内，意外获得亲和力提升的优势人源化单抗，亲和力较阳性对照抗体83A10提升10％-90％。同时种属交叉特性保留，与猴结合的亲和力较人鼠嵌合单抗有显著提高。
实施例12 BiaCore法测定人源化单克隆抗体的亲和力
在本实施例中，使用BIACORE方法测定抗原-抗体结合动力学及亲和力。
试验材料：人BCMA/TNFRSF17蛋白(Human BCMA/TNFRSF17 Protein)(ACRO，BCA-H522y)；食蟹猴/恒河猴BCMA/TNFRSF17蛋白(Cynomolgus/Rhesus macaque BCMA/TNFRSF 17Protein)(ACRO，BCA-C52H7)；Sereis S Sensor Chip CM5(GE，BR100530)；抗组氨酸抗体(Anti-histidine antibody)(GE，28995056)；HBS-EP(10X)(GE，BR-1006-69)；甘氨酸10mM，pH 1.5(GE，BR100354)。
试验方法：将抗组氨酸抗体(GE，His捕获试剂盒(His capture Kit)，货号：28995056)偶联至Sereis S Sensor Chip CM5芯片上，捕获供试品，抗原作为分析物检测其与供试品结合的动力学和亲和力数据。抗原与供试品结合的检测起始浓度为10nM，在此基础上，2倍梯度稀释，即抗原稀释的浓度分别为10nM、5nM、2.5nM、1.25nM、0.625nM，抗原依次从低浓度到高浓度开始进样，并设置1个阴性对照(即 1×HBS-EP+buffer)和1个重复浓度(一般为最低浓度重复)，进样前至少进行3次Start up(1×HBS-EP+buffer)的冲洗流程以平衡***，检测抗原与供试品的结合和解离趋势，当解离完成后，再生试剂进样，对芯片进行再生，芯片再生完成后，进行下一个浓度的检测。检测完成后，利用数据分析软件(Biacore T200 Evaluation Software)中的1∶1 Binding拟合方式进行数据拟合。试验结果见表14。
表14：人源化抗体对人BCMA及猴BCMA亲和力检测结果
Figure PCTCN2020136748-appb-000011
与人/猴BCMA的亲和力检测结果显示，本发明抗BCMA的人源化抗体的亲和力比对照抗体83A10(专利WO2014122144A1)高一至二个数量级，具有更强的亲和力。
实施例13：人源化抗体抑制APRIL配体与BCMA的结合
APRIL结合竞争ELISA评价抗-BCMA人源化抗体阻断APRIL与BCMA抗原结合的能力。
试验材料：Biotin标记试剂盒(DoJindo，LK03)，可溶性人APRIL(ACRO，货号APL-H5244)，cynoBCMA-his(ACRO，BCA-C52H7)和huBCMA-his(ACRO，BCA-H522y)，TMB Substrate Reagent Set(BD，555214)。
试验方法：同实施例7，用人源化单抗替代人鼠嵌合单抗。
表15：人源化抗体竞争抑制APRIL配体结合人BCMA活性比较
样品 IC50(ng/mL)
APRIL 3700
hu-mAb1 1646
hu-mAb2 2758
hu-mAb3 918.4
hu-mAb4 476.4
试验结果如表15和图7所示。结果证明，人源化抗体hu-mAb1、hu-mAb2、 hu-mAb3和hu-mAb4能够抑制APRIL与huBCMA-his的结合且趋势与嵌合抗体一致。当抗体浓度为12ug/mL时，hu-mAb1和hu-mAb2的抑制率分别为70％和60％。hu-mAb3和hu-mAb4与huBCMA-his有较高的亲和力，但未能显著抑制APRIL与huBCMA-his的结合，此结果表明抗体hu-mAb3和hu-mAb4仍可能具备不同于APRIL与huBCMA-his的结合位点。
实施例14 人源化抗体抑制阳性抗体83A10结合
试验方法同实施例8，用人源化单抗替代人鼠嵌合单抗。
表16：人源化抗体竞争抑制抗体83A10结合人和猴BCMA活性比较
Figure PCTCN2020136748-appb-000012
试验结果如表16、图8和9所示。结果证明，人源化抗体能够抑制83A10与huBCMA-his的结合且趋势与嵌合抗体一致。当抗体浓度为7μg/mL时，hu-mAb1和hu-mAb2的抑制率分别为60％和30％。hu-mAb3和hu-mAb4与huBCMA-his有较高的亲和力，但未能显著抑制83A10与huBCMA-his的结合，此结果表明抗体hu-mAb3和hu-mAb4仍可能具备不同于APRIL与huBCMA-his的结合位点。
实施例15 人源化抗体的细胞亲和力评估
使用FACS方法评估4株人源化单抗与细胞表面BCMA抗原的亲和力。
试验材料：细胞株NCI-H929，HEK293 cynoBCMA，HEK293；缓冲液：1％FBS-PBS，pH 7.4；PE抗人IgG Fc(Biolegend，409304)
试验方法：制备细胞悬液：用PBS重悬细胞，调整细胞浓度，使每孔细胞数量为2×10 5。在96孔板中加入待检鼠源抗体50μL，抗体起始浓度3000nM，3倍梯度稀释，4℃孵育1小时，一抗孵育结束后，向96孔板中加入150μL预冷的含1％FBS-PBS，300×g离心5min后去上清，重复上述操作1次。加入荧光二抗PE抗人IgG Fc(Biolegend，409304)，每孔80μL，4℃孵育30分钟，二抗孵育结束后，向96孔板中加入150μL预冷的含1％FBS PBS，将96孔板放入板式离心机中配平，300×g离心5min后去上清，重复上述操作1次。100μL含1％FBS PBS重悬，流式细胞仪(BD Accuri TM C6)检测。试验结果如表17所示。
表17：人源化抗体与人/猴BCMA表达细胞株的结合活性比较
Figure PCTCN2020136748-appb-000013
结果表明，c-mAb1，c-mAb2和c-mAb3人源化后对应的人源化抗体亲和力变化在3倍以内。c-mAb1，c-mAb2和c-mAb3及对应人源化单抗，hu-mAb1，hu-mAb2，hu-mAb3和hu-mAb4与NCI-H929细胞株的亲和力均优于83A10；具有人猴交叉的母抗体c-mAb3及其人源化单抗hu-mAb3和hu-mAb4均具有人猴交叉性，且三者与HEK293 cynoBCMA的亲和力也优于83A10。
实施例16 人源化抗体的ADCC作用
评估人源化前后抗体介导ADCC强弱评估。
试验材料：细胞株NCI-H929，U266B1，NK92MI-CD16a(宜明昂科生物医药技术(上海)有限公司)；PBS缓冲液；羟基荧光素二醋酸盐琥珀酰亚胺脂(5，6-carboxyfluorescein diacetate，succinimidyl ester，CFSE，eBioscience，65-0850-84)；溴化丙锭(Propidium iodide，PI，Sigma，P4170)。
试验方法：取靶细胞NCI-H929，U266B1和效应细胞NK92MI-CD16a进行细胞计数检测测活力，取待使用细胞进行300×g离心5min收集细胞并用5μM CFSE将细胞染色(37℃，15min)，完全培养基洗两次后于VI-Cell细胞计数仪(Beckman)上计数，后按照实验设计加入96孔板中，每孔2×10 4个细胞/100μL。按照50μL/孔加入配制好的4×抗体分子，将NK92MI-CD16a加入96孔板中(每孔1×10 5个细胞/50μL，效靶比为5∶1)。将细胞培养板置于细胞培养箱中培养6h后取出加入终浓度为1μg/mL的PI，孵育10min后用流式细胞仪(BD Accuri TM C6)上机检测，并分析CFSE+PI+双阳性细胞占CFSE+阳性细胞百分比。
实验结果如图10和图11所示，对于BCMA阳性靶细胞，本发明嵌合抗体c-mAb1，c-mAb2和c-mAb3和人源化单抗hu-mAb1，hu-mAb2，hu-mAb3和hu-mAb4均具有显著的ADCC作用。并且，在效靶比5∶1，0.1pM抗体浓度处理NCI-H929，人源化单抗较母本抗体提高20％-70％；在效靶比5∶1，100pM抗体浓度处理U266B1，人源化单抗较母本抗体提高20％-120％。

Claims (17)

  1. 抗BCMA抗体或其抗原结合片段，其特征在于包含
    (i)SEQ ID NO：1所示重链可变区包含的HCDR1，HCDR2和HCDR3；和/或SEQ ID NO：2所示轻链可变区包含的LCDR1，LCDR2和LCDR3：
    (ii)SEQ ID NO：3所示重链可变区包含的HCDR1，HCDR2和HCDR3；和/或SEQ ID NO：4所示轻链可变区包含的LCDR1，LCDR2和LCDR3：
    (iii)SEQ ID NO：5所示重链可变区包含的HCDR1，HCDR2和HCDR3；和/或SEQ ID NO：6所示轻链可变区包含的LCDR1，LCDR2和LCDR3：或
    (iv)重链可变区包含的HCDR1，HCDR2和HCDR3；和/或轻链可变区包含的LCDR1，LCDR2和LCDR3；其中，所述重链可变区和/或轻链可变区与(i)至(iii)任一所述的重链可变区和/或轻链可变区相比，至少一个CDR含有突变，所述突变为一个或多个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)并能够保留与BCMA结合亲和力；优选地，所述的突变为保守氨基酸突变；
    优选地，按照IMGT、Kabat、Chothia或AbM编号***定义；优选地，按照IMGT编号***，所述抗BCMA抗体包含以下CDRs，
    (1)HCDR1，其包含SEQ ID NO：7所示的氨基酸序列或其变体，或由其组成，
    HCDR2，其包含SEQ ID NO：8所示的氨基酸序列或其变体，或由其组成，
    HCDR3，其包含SEQ ID NO：9所示的氨基酸序列或其变体，或由其组成，
    LCDR1，其包含SEQ ID NO：16所示的氨基酸序列或其变体，或由其组成，
    LCDR2，其包含SEQ ID NO：17所示的氨基酸序列或其变体，或由其组成，和
    LCDR3，其包含SEQ ID NO：18所示的氨基酸序列或其变体，或由其组成；
    (2)HCDR1，其包含SEQ ID NO：10所示的氨基酸序列或其变体，或由其组成，
    HCDR2，其包含SEQ ID NO：11所示的氨基酸序列或其变体，或由其组成，
    HCDR3，其包含SEQ ID NO：12所示的氨基酸序列或其变体，或由其组成，
    LCDR1，其包含SEQ ID NO：19所示的氨基酸序列或其变体，或由其组成，
    LCDR2，其包含SEQ ID NO：20所示的氨基酸序列或其变体，或由其组成，和
    LCDR3，其包含SEQ ID NO：21所示的氨基酸序列或其变体，或由其组成；或
    (3)HCDR1，其包含SEQ ID NO：13所示的氨基酸序列或其变体，或由其组成，
    HCDR2，其包含SEQ ID NO：14所示的氨基酸序列或其变体，或由其组成，
    HCDR3，其包含SEQ ID NO：15所示的氨基酸序列或其变体，或由其组成，
    LCDR1，其包含SEQ ID NO：22所示的氨基酸序列或其变体，或由其组成，
    LCDR2，其包含SEQ ID NO：17所示的氨基酸序列或其变体，或由其组成，和
    LCDR3，其包含SEQ ID NO：23所示的氨基酸序列或其变体，或由其组成，
    其中，所述变体为与序列编号所示的对应CDR序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性并保留与BCMA结合亲和力的序列，或与序列编号所示的对应CDR序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，***或缺失)并保留与BCMA结合亲和力的氨基酸序列；
    优选地，所述抗体或其抗原结合片段的重链可变区和/或轻链可变区包含来自人源、鼠源或兔源的FR区。
  2. 权利要求1所述的抗BCMA抗体或其抗原结合片段，其中所述抗体的重链可变区包含下述序列或其变体，或由下述序列或其变体组成：SEQ ID NO：1、SEQ ID NO：3、SEQ ID NO：5、SEQ ID NO：24、SEQ ID NO：26、SEQ ID NO：28或SEQ ID NO：30；并且
    所述抗体的轻链可变区包含下述序列或其变体，或由下述序列或其变体组成：SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：25、SEQ ID NO：27、SEQ ID NO：29或SEQ ID NO：31；
    其中所述变体为与序列编号所示的对应的抗体重链可变区或轻链可变区的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性并保留与BCMA结合亲和力的序列，或与序列编号所示的对应的抗体重链可变区或轻链可变区的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，***或缺失)并保留与BCMA结合亲和力的氨基酸序列。
  3. 权利要求1或2所述的抗BCMA抗体或其抗原结合片段，其中
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：1或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：2或其变体所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：3或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：4或其变体所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：5或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：6或其变体所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：24或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：25或其变体所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：26或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：27或其变体所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：28或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：29或其变体所示；或
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：30或其变体所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：31或其变体所示。
  4. 权利要求1或2所述的抗BCMA抗体或其抗原结合片段，其中所述抗体的重链恒定区和所述抗体的轻链恒定区来自人IgG1，IgG2，IgG3或IgG4，优选为人IgG1重链恒定区，GenBank ACCESSION：AK303185.1；轻链恒定区为Ig1 kappa链恒定区，GenBank ACCESSION：MG815648.1。
  5. 权利要求1或2所述的抗BCMA抗体或其抗原结合片段，其中所述抗体为单克隆抗体、部分或全人源化抗体、嵌合抗体、单链抗体、多特异性抗体(例如双特异性抗体)。
  6. 权利要求1或2所述的抗BCMA抗体或其抗原结合片段，所述抗原结合片段选自Fab、Fab′、F(ab′) 2、F(ab) 2、Fd、Fv、dAb、Fab/c、互补决定区片段、scFv、scFv多聚体、二硫键稳定性Fv(dsFv)、(dsFv) 2、双特异性dsFv(dsFv-dsFv′)、双链抗体(Diabody)、二硫键稳定的双链抗体(ds-Diabody)、由包含一个或多个CDR的抗体的一部分形成的多特异性抗体、单域抗体(sdab)、纳米抗体、结构域抗体或二价结构域抗体。
  7. 分离的多肽或其变体，其选自以下各项组成的组：
    (1)分离的多肽或其变体，其包含SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的序列；
    (2)分离的多肽或其变体，其包含SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的序列；
    (3)分离的多肽或其变体，其包含SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：21所示的序列；
    (4)分离的多肽或其变体，其包含SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：21所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的序列；
    (5)分离的多肽或其变体，其包含SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：22、SEQ ID NO：17和SEQ ID NO：23所示的序列；
    (6)分离的多肽或其变体，其包含SEQ ID NO：22、SEQ ID NO：17和SEQ ID NO：23所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还应包含SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的序列；
    (7)分离的多肽或其变体，其包含SEQ ID NO：1所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：2所示的序列；
    (8)分离的多肽或其变体，其包含SEQ ID NO：2所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：1所示的序列；
    (9)分离的多肽或其变体，其包含SEQ ID NO：3所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：4所示的序列；
    (10)分离的多肽或其变体，其包含SEQ ID NO：4所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：3所示的序列；
    (11)分离的多肽或其变体，其包含SEQ ID NO：5所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：6所示的序列；
    (12)分离的多肽或其变体，其包含SEQ ID NO：6所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：5所示的序列；
    (13)分离的多肽或其变体，其包含SEQ ID NO：24所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：25所示的序列；
    (14)分离的多肽或其变体，其包含SEQ ID NO：25所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：24所示的序列；
    (15)分离的多肽或其变体，其包含SEQ ID NO：26所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：27所示的序列；
    (16)分离的多肽或其变体，其包含SEQ ID NO：27所示的序列，其中所述多 肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：26所示的序列；
    (17)分离的多肽或其变体，其包含SEQ ID NO：28所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：29所示的序列；
    (18)分离的多肽或其变体，其包含SEQ ID NO：29所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：28所示的序列；
    (19)分离的多肽或其变体，其包含SEQ ID NO：30所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：31所示的序列；或
    (20)分离的多肽或其变体，其包含SEQ ID NO：31所示的序列，其中所述多肽作为抗BCMA抗体的一部分，特异性结合BCMA，所述抗体还包含SEQ ID NO：30所示的序列，
    其中所述变体为与序列编号所示的对应序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性并保留与BCMA结合亲和力的序列，或与序列编号所示的对应序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，***或缺失)并保留与BCMA结合亲和力的氨基酸序列。
  8. 核酸分子，其编码权利要求1-6任一项所述的抗体或其抗原结合片段，或权利要求7所述分离的多肽或其变体。
  9. 载体，其包含权利要求8所述的核酸分子。
  10. 宿主细胞，其包含权利要求8所述的核酸分子，或权利要求9所述的载体。
  11. 偶联物，其包含权利要求1-6任一项所述的抗体或其抗原结合片段以及偶联部分，其中，所述偶联部分为纯化标签(如His标签)、可检测的标记、药物、毒素、细胞因子、酶、或其组合；优选地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质、化疗剂、生物毒素、聚乙二醇或酶。
  12. 融合蛋白或多特异性抗体(优选双特异性抗体)，其包含权利要求1-6任一项所述的抗体或其抗原结合片段；优选地，所述融合蛋白为CAR构建物，所述CAR构建物特异性结合BCMA。
  13. 试剂盒，其包括权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体；优选地，所述试剂盒还包括第二抗体，其特异性识别所述抗体；任选地，所述第二抗体还 包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；优选地，所述试剂盒用于检测BCMA在样品中的存在或其水平。
  14. 药物组合物，其包含权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体；可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂；优选地，所述药物组合物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
  15. 权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体用于治疗和/或预防肿瘤(例如多发性骨髓瘤)，或在制备治疗和/或预防肿瘤(例如多发性骨髓瘤)的药物中的用途，或者在制备用于诊断肿瘤的药物中的用途。
  16. 试剂盒，其包含(1)权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体，和(2)针对其它抗原(如CD38和/或SLAMF7)的抗体或其抗原结合片段，和/或细胞毒性剂，和任选地，使用说明书。
  17. 治疗或预防肿瘤的(例如多发性骨髓瘤)方法，包括向受试者施用治疗有效量的权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体。
PCT/CN2020/136748 2020-12-16 2020-12-16 抗bcma抗体及其制备方法和应用 WO2022126416A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US18/257,749 US20240117061A1 (en) 2020-12-16 2020-12-16 Anti-bcma antibody, preparation method therefor and application thereof
PCT/CN2020/136748 WO2022126416A1 (zh) 2020-12-16 2020-12-16 抗bcma抗体及其制备方法和应用
CN202080107895.2A CN116783220A (zh) 2020-12-16 2020-12-16 抗bcma抗体及其制备方法和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/136748 WO2022126416A1 (zh) 2020-12-16 2020-12-16 抗bcma抗体及其制备方法和应用

Publications (1)

Publication Number Publication Date
WO2022126416A1 true WO2022126416A1 (zh) 2022-06-23

Family

ID=82059861

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/136748 WO2022126416A1 (zh) 2020-12-16 2020-12-16 抗bcma抗体及其制备方法和应用

Country Status (3)

Country Link
US (1) US20240117061A1 (zh)
CN (1) CN116783220A (zh)
WO (1) WO2022126416A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012066058A1 (en) * 2010-11-16 2012-05-24 Boehringer Ingelheim International Gmbh Agents and methods for treating diseases that correlate with bcma expression
CN103509113A (zh) * 2007-07-12 2014-01-15 皮埃尔法布雷医药公司 抑制c-MET二聚化的新型抗体及其用途
CN109485733A (zh) * 2018-12-28 2019-03-19 广州百暨基因科技有限公司 一种全人源的抗bcma嵌合抗原受体及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103509113A (zh) * 2007-07-12 2014-01-15 皮埃尔法布雷医药公司 抑制c-MET二聚化的新型抗体及其用途
WO2012066058A1 (en) * 2010-11-16 2012-05-24 Boehringer Ingelheim International Gmbh Agents and methods for treating diseases that correlate with bcma expression
CN109485733A (zh) * 2018-12-28 2019-03-19 广州百暨基因科技有限公司 一种全人源的抗bcma嵌合抗原受体及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN HAIMING; LI MINGJIE; XU NING; NG NICOLE; SANCHEZ ERIC; SOOF CAMILIA M.; PATIL SAURABH; UDD KYLE; BUJARSKI SEAN; CAO JASMIN; H: "Serum B-cell maturation antigen (BCMA) reduces binding of anti-BCMA antibody to multiple myeloma cells", LEUKEMIA RESEARCH, NEW YORK,NY, US, vol. 81, 1 January 1900 (1900-01-01), US , pages 62 - 66, XP085692583, ISSN: 0145-2126, DOI: 10.1016/j.leukres.2019.04.008 *
DATABASE PROTEIN 10 March 2016 (2016-03-10), ANONYMOUS : "immunoglobulin heavy chain variable region, partial [Mus musculus] ", XP055943656, retrieved from NCBI Database accession no. AML31737 *
DATABASE PROTEIN 24 July 2016 (2016-07-24), ANONYMOUS : "anti-glycyrrhetic acid antibody GA129 light chain, partial [Mus musculus]", XP055943660, retrieved from NCBI Database accession no. BAC56969 *

Also Published As

Publication number Publication date
CN116783220A (zh) 2023-09-19
US20240117061A1 (en) 2024-04-11

Similar Documents

Publication Publication Date Title
CN110382536B (zh) 抗pd-1抗体及其用途
US11555077B2 (en) 4-1BB antibody and preparation method and use thereof
TWI673287B (zh) 抗b7-h3抗體、其抗原結合片段及其醫藥用途
CN110267989B (zh) 抗cd40抗体、其抗原结合片段及其医药用途
EP3753951A1 (en) Anti-b7-h4 antibody, antigen-binding fragment thereof and pharmaceutical use thereof
CN112969714B (zh) 抗cd40抗体、其抗原结合片段及其医药用途
KR20190112299A (ko) 결합제
IL301956A (en) Antibodies against dectin-1 and methods of using them
WO2021143914A1 (zh) 一种激活型抗ox40抗体、生产方法及应用
CN115298216A (zh) 抗体或其抗原结合片段、其制备方法及医药用途
US11542329B2 (en) Antibodies targeting Glycoprotein VI
EP4302778A1 (en) Pharmaceutical composition containing anti-tslp antibody
WO2022126416A1 (zh) 抗bcma抗体及其制备方法和应用
RU2779128C2 (ru) Антитело к cd40, его антигенсвязывающий фрагмент и его медицинское применение
WO2022012639A1 (zh) Pd-1抗原结合蛋白及其应用
CA3210910A1 (en) Anti-pd-l1 antibody and use thereof
CN116478288A (zh) 红细胞弱结合型人源化cd47抗体及其应用
IL310427A (en) Pharmaceutical preparation of anti-R4IL antibody and use thereof
CN116496404A (zh) 靶向cd47和pd-l1的双特异性抗体及其应用
CN113811545A (zh) 对btn2具有特异性的抗体及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20965434

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202080107895.2

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 18257749

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20965434

Country of ref document: EP

Kind code of ref document: A1